Compositions and Methods for the Treatment of Inflammation in Urological Pathology

ABSTRACT

The present disclosure provides compositions and methods for the treatment of inflammation in urological pathologies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/850,015, filed May 20, 2019, the disclosure of which is herebyincorporated by reference in its entirety.

FEDERAL FUNDING LEGEND

This invention was made with Government support under Federal Grant Nos.R01DK103534 and R01DK117890 awarded by the National Institutes ofHealth. The Federal Government has certain rights to this invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

This application contains a Sequence Listing submitted as an electronictext file named “20-781-US_SequenceListing_ST25.txt,” having a size inbytes of 3 kb, and created on May 20, 2020. The information contained inthis electronic file is hereby incorporated by reference in itsentirety.

BACKGROUND Description of Related Art

Inflammasomes are supramolecular complexes that were discovered in 2002and found to be of central importance in initiating inflammation inresponse to sterile as well as infectious stimuli. Nod-like receptorNLRP3 inflammasome, a subset type of inflammasome, is the best studiedof the NOD-like receptor (NLR) family of pattern receptors. The NLRP3inflammasome is comprised, in part, of the nod-like receptor NLRP3, astructural co-factor protein called thioredoxin-interacting protein(TXNIP), and the adaptor protein Apoptosis-Associated Speck-like ProteinC (ASC).

In general, pattern receptors recognize molecules released from damagedor dying cells (or those with deranged metabolism), known as damage (ordanger) associated molecular patterns (DAMPS) or components of pathogensknown as pathogen associated molecular patterns (PAMPS). NLRP3 is thebest understood NLR to recognize DAMPS and has been implicated in manydiseases with a sterile inflammatory component, including diabeticcomplications. Upon recognition of DAMPS, NLRP3 oligomerizes andtriggers enucleation of ASC. ASC in turn interacts with procaspase-1which is cleaved and activated through an auto-proteolytic process.Caspase-1, in turn, catalyzes the enzymatic maturation IL-1β, IL-18 andgasdermin D. Gasdermin D forms a pore in the plasma membrane, triggeringa programmed necrosis called pyroptosis which releases IL-1β and IL-18that act as proinflammatory cytokines to initiate the inflammatoryresponse.

NLRP3 has been shown to play an important role in the urinary tract ofthe rodent. (Inouye et al. (2018) Curr. Urol. 11:57-72; Purves et al.(2016) Am. 1 Physiol. Renal Physiol. 311:F653-F662). In the rat bladder,NLRP3 is localized to the urothelium (Hughes et al. (2015) Int. Urol.Nephrol. 47:1953-1964; Hughes et al. (2014) Am. I Physiol. RenalPhysiol. 306:F299-308) where it mediates sterile inflammation in severalimportant bladder pathologies including bladder outlet obstruction andcyclophosphamide-induced hemorrhagic cystitis. (Id.; Hughes et al.(2016) J. Urol. 195:1598-1605). Experimental models have also implicatedNLRP3 in the response to urinary tract infections. (Hughes et al. (2016)J. Clin. Cell Immunol. 7(1):396; Hamilton et al. (2017) Nature Rev.Urology 14:284-295).

NLRP3 is also implicated in inflammatory processes in diabetic patients.Indeed, it is now appreciated that diabetes is not just a disease ofhigh blood sugar but also a disease of deranged metabolism resulting inhyperglycemia and the production of numerous metabolites such as uricacid and free fatty acids, where these metabolites trigger inflammationthat damages susceptible tissues with a resulting loss of function.(Shin et al. (2015) Ageing Res. Rev. 24:66-76; Hameed et al. (2015)World I Diabetes 6:598-612). Recent breakthroughs in certain diabeticcomplications (nephropathy, retinopathy, and cardiomyopathy) havedemonstrated that this inflammation results from activation of NLRP3.(Sepehri et al. (2017) Immunol. Lett. 192:97-103). However, the role ofNLRP3 in urological disorders associated with diabetes, such as diabeticbladder dysfunction (DBD), has not previously been established. DBDaffects up to 87% of diabetic patients, and there are currently notargeted therapies for DBD. (Daneshgari et al. (2006) Semin. Nephrol.26:182-185; Panigrahy et al. (2017) Diabetes Metab. Syndr. 11:81-82).

Other diseases and disorders of urinary and bladder dysfunction involve,at least in part, inflammation in urological pathology. Urine containsmany noxious chemical moieties, such as organic acids and salts, whichcan irritate or inflame the lumen of the urinary tract. In patients whoare particularly sensitive to these, including those who have defects inthe protective glycosaminoglycan (GAG) layer, they can cause urinarysymptoms of urgency and frequency or even pain. The most potent forms ofthese irritants are the components that can crystallize into urinarystones. Once they have crystallized to a sufficiently large size, theyare capable of mechanically breaching the protective GAG layer to exposethe vulnerable urothelium below. Perhaps the most extreme form of thisirritation is seen when stone material becomes impacted in the ureterand can cause intense pain from renal colic and local inflammation.Moreover, impacted ureteral stones produce local fibrosis and are themost common known cause of ureteral strictures that can cause long-termmorbidity and need for surgical intervention. (Dong et al. (2018) AsianJ Urol. 5(2):94-100; Roberts et al. (1998) J. Urol. 159(3):723-726).Exactly how these chemical urinary components interface with theurothelium to provoke functional disturbances, fibrosis, and pain is notcompletely understood.

The consequences of urothelial exposure to noxious chemical stimuli arethought to potentially result from inflammation. Recent studies ofinflammation in innate immune cells and epithelial tissues haveidentified the inflammasome as being critically important in mediatingthis inflammatory response. (Savage et al. (2012) Frontiers Immunol.3:288; Rheinheimer et al. (2017) Metabolism. 74:1-9; Liu et al. (2018)Basic Res. Cardiol. 113(1):5). In studies of gout and pseudogout(Martinon et al. (2006) Nature. 440(7081):237-241), calciumpyrophosphate (CPPD) and monosodium urate (MSU), two major components ofurinary stones, have been shown to act as DAMPs to stimulate NLRP3inflammasome activity in macrophages. In similar studies ofosteoarthritis, both CPPD and MSU promote inflammation mediated by thisinflammasome. (Campillo-Gimenez et al. (2018) Frontiers in Immunol.9:2248). In their role as DAMPs, these two components are thought topotentiate NLRP3 inflammasome activation by stimulating intracellularreactive oxygen species (ROS) production. Under normal conditions, TXNIPis bound to the cellular antioxidant thioredoxin. When ROS are produced,they oxidize thioredoxin, resulting in the dissociation of TXNIP. FreeTXNIP is then able to bind to NLRP3 to promote formation of the activeinflammasome. While ROS-mediated NLRP3 activation, and the importance ofTXNIP, have been explored in other cell types (Joshi et al. (2015) J.Urol. 193(5):1684-1691; Minutoli et al. (2016) Oxid. Med. Cell Longev.2016:2183026), its role in stone-mediated urothelial inflammation hasnot previously been defined.

Moreover, in certain instances, symptoms associated with urologicaldiseases and disorders are not limited to the urinary tract. Forexample, there are numerous accounts in the literature of theassociation between Lower Urinary Tract Symptoms (LUTS) and depression.(Coyne et al. (2009) BJU Intl. 103 Suppl 3: 4-11). Particularlypersuasive are the conclusions of the EpiLUTS study (Epidemiology ofLUTS study) study (Id.; Milsom et al. (2012) Urology 80: 90-96),although mood disorders have been anecdotally associated with many ofthe underlying diseases for years including recurrent urinary tractinfections (Renard et al. (2014) Infect. Dis. Ther. 4(1):125-135),overactive bladder (Golabek et al. (2016) Psychiatr. Pol. 50: 417-430;Lai et al. (2015) BMC Urol. 15: 14; Tzeng et al. (2019) J. Investig.Med. 67(2):312-318; Wu et al. (2017) Front Cell Infect. Microbiol.7:488), bladder outlet obstruction (Dunphy et al. (2015) Rev. Urol.17:51-57) and incontinence (Giannantoni et al. (2018) J. Urol.199:e350-e351). Probably best known for this association is interstitialcystitis (IC)/bladder pain syndrome, a prevalent condition affecting upto 8 million women in the United States (Berry et al. (2011) J Urol.186: 540-544) that is strongly associated with depression (Hepner et al.(2012) Urology 80: 280-285; McKernan et al. (2018) Neurourology andUrodynamics 37: 926-941; Meijlink J M. (2017) Urologia 84: 5-7; Muere etal. (2018) Pain Manag. Nurs. 19(5):497-505) and suicidal ideation(Hepner et al. (2012) Urology 80: 280-285). Despite considerable effortto understand the origin of these symptoms, the etiology has remainedenigmatic.

Recently, breakthroughs have shown that several acute and chronicdiseases of peripheral tissues trigger inflammation in the centralnervous system (CNS). (Hamasaki et al. (2018) J Neurosci. Res. 96:371-378). Much of this groundbreaking knowledge is derived from studiesof the gastrointestinal system. For example, Hsieh et al. demonstratedthat as little as 30 minutes of ischemia in the intestines results in anincrease in expression of inflammatory mediators and activation ofmicroglia within the CNS. (Hsieh et al. (2011) Shock 36: 424-430). Inaddition, irritable bowel syndrome, which might be considered a colonicparallel to interstitial cystitis due to its unknown origin,inflammatory nature, and similar psychosocial comorbidities, alsotriggers neuroinflammation. (Daulatzai Mass. (2014) Neurochem. Res. 39:624-644). Importantly, the conditions peripheral to central pathways inquestion are not limited to the gastrointestinal system, for otherperipheral insults such as burns, cardiac arrest, and acute pancreatitiscan all result in CNS inflammation. (Hamasaki et al. (2018) J NeurosciRes 96: 371-378). Neuroinflammation is well-known to cause mooddisorders (Miller et al. (2009) Biol. Psychiatry 65:732-741; Miller andRaison (2016) Nature Rev. Immunol. 16:22-34; Noto et al. (2014)Neuroimmunomodulation 21:131-139; Sayana et al. (2017) J Psychiatr. Res.92:160-182; Teixeira and Muller (2014) Neuroimmunomodulation 21:71) andplays a major role in debilitating diseases such as major depressivedisorder and bipolar disorder (Colpo et al. (2018) Expert Rev.Neurother. 18: 139-152; Haroon et al. (2017) Neuropsychopharmacology 42:193-215).

Clearly, the evidence suggests peripheral insults can triggerneuroinflammation and neuroinflammation can cause pain, but it has beenfurther hypothesized that neuroinflammation may continue to alter normalphysiology following the resolution of local inflammation, such aspersistent discomfort in the bladder following an acute or recurrentinfection and it may do so by changing nociceptive thresholds. Thus,neuroinflammation may explain residual symptoms. Equally important,neuroinflammation has been implicated in the development of mooddisorders, including major depressive disorder and generalized anxietydisorder, which are prevalent co-morbid conditions in patients withchronic pain syndromes such as IC. However, whether insults to thebladder may result in neuroinflammation in the CNS that leads to thepsychosocial symptoms, or the underlying mechanism under which thismight occur, have not previously been established or understood.

BRIEF SUMMARY OF THE DISCLOSURE

This Summary is provided to introduce a selection of concepts that arefurther described below in the Detailed Description. This Summary is notintended to identify key or essential features of the claimed subjectmatter, nor is it intended to be used as an aid in limiting the scope ofthe claimed subject matter.

One aspect of the present disclosure provides a method of treatinginflammation in the bladder in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of aninflammasome inhibitor.

In some embodiments of the disclosure, the inflammation in the bladderis an acute inflammation or a chronic inflammation.

In some embodiments of the disclosure, the inflammation in the bladderis induced by a danger associated molecular pattern (DAMP) or a pathogenassociated molecular pattern (PAMP). In some embodiments of thedisclosure, the DAMP is ATP, calcium pyrophosphate (CPPD), monosodiumurate (MSU), high mobility group box-1 (HMG-B1), albumin, uromodulin,uric acid crystals, hypoxia, acrolein, calcium oxalate, cholesterol,reactive oxidative species (ROS) serum amyloid A (SAA), amyloid βfibril, hyaluronan, aluminum, asbestos, silica, UV radiation, drusen, orskin irritants.

In some embodiments of the disclosure, the PAMP is a fungus (e.g.,Candida albicans, Saccharomyces cerevisiae, or Aspergillus fumigatus),bacteria (e.g., Listeria monocytogenes, Staphylococcus aureus,Escherichia coli, Chlamydia pneumonia, Mycobacterium tuberculosis,Clostridium difficile, Bordetella pertussis, Vibrio cholera, Neisseriagonorrhoeae, or Streptococcus pyogenes), or virus (e.g., Influenza A,adenovirus, Sendai virus, Varicella-zoster, or herpes).

In some embodiments of the disclosure, the inflammation in the bladdercomprises urothelial cell damage.

In some embodiments of the disclosure, the subject is a human.

In some embodiments of the disclosure, the inflammasome inhibitor is anNLRP1 inflammasome inhibitor, an NLRP3 inflammasome inhibitor, an NLRP6inflammasome inhibitor, an NLRP7 inflammasome inhibitor, an NLPR9inflammasome inhibitor, an NLRP12 inflammasome inhibitor, an NLRC4inflammasome inhibitor, or an AIM2 inflammasome inhibitor.

In other embodiments of the disclosure, the inflammasome inhibitor is anNLRP3 inflammasome inhibitor. In some embodiments of the disclosure, theNLRP3 inflammasome inhibitor (e.g., glyburide). In other embodiments ofthe present disclosure, the NLRP3 inflammasome inhibitor is a TXNIPinhibitor (e.g., verapamil), ASC inhibitor, NEK7 inhibitor, Gasdermin Dinhibitor, capspase-11 inhibitor, capsase-1 inhibitor (e.g., verapamil),IL-1β inhibitor, IL-18 inhibitor or combinations thereof. In otherembodiments of the present disclosure, the NLRP3 inflammasome inhibitoris a ROS scavenger (e.g., N-acetylcysteine (NAC)).

In some embodiments of the disclosure, the subject is diagnosed withdiabetes, a urinary tract infection, urinary frequency, fibrosis,bladder outlet obstruction (BOO), interstitial cystitis, CP-inducedcystitis, depression, anxiety, neuroinflammation, a gynecologic cancer,kidney stones, a pelvic inflammatory disorder, endometriosis, Chron'sdisease, diverticulitis, lupus, tuberculosis, and combinations thereof.

In some embodiments of the present disclosure, the subject had beenexposed to chemotherapy, radiation, a catheter, or a urinary stent.

Another aspect of the present disclosure provides a method of treatingdiabetic bladder dysfunction (DBD) in a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of an inflammasome inhibitor.

Yet another aspect of the present disclosure provides a method oftreating or preventing a condition associated with neuroinflammation,the method comprising administering a therapeutically effective amountof an inflammasome inhibitor.

In some embodiments of the present disclosure, the subject that has beendiagnosed with inflammation of the bladder or an inflammatory bladderdisorder. In some embodiments of the disclosure, the inflammatorybladder disorder is interstitial cystitis, BOO, or DBD.

In some embodiments of the present disclosure, the condition associatedwith neuroinflammation is a mood disorder in the subject (e.g,depression, dysthymic disorder, bipolar disorder, anxiety, or anhedonia,or combinations thereof).

In some embodiments of the disclosure, the method further comprisesadministering a therapeutically effective amount of an antidepressantagent. In some embodiments of the disclosure, the antidepressant agentis selected from the group consisting of a selective serotonin reuptakeinhibitors (SSRIs), a norepinephrine-dopamine reuptake inhibitors(NDRIs), or a monoamine oxidase inhibitors (MAOIs). In some embodiments,the antidepressant agent is fluoxetine.

Yet another aspect of the present disclosure provides a new murine modelof diabetic mice lacking the NLRP3 inflammasome (NLRP3^(−/−), diab).

Yet another aspect of the present disclosure provides a compositioncomprising an inflammasome inhibitor and a pharmaceutically acceptablecarrier or excipient.

Additional features and advantages are described herein, and will beapparent from the following Detailed Description, Drawings, and theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and other features of the disclosure are explainedin the following description, taken in connection with the accompanyingdrawings, wherein:

FIGS. 1A-1G show analysis of various DAMPS on inflammasome activation inurothelial cells in vitro. Cells were isolated, plated and treated withthe doses of the various compounds indicated. Following incubationperiods described below, caspase-1 was measured. FIG. 1A is a graphshowing ATP dose-response. Each point represents the mean±SEM. n=5 foreach dose. Asterisks indicate significant differences from 0 mM ATPcontrol. ***p<0.001 by ANOVA followed by Tukey's post-hoc test. FIG. 1B.is graph showing the effects of LPS on the ATP dose-response. Urothelialcells were plated for 24 h, then LPS (1 μg/ml) in 10 μl PBS (or PBSalone) added for an additional 24 h. Then the indicated doses of ATPwere added for 1 h prior to caspase-1 analysis. The −LPS samples (closedtriangles) are the exact same samples shown in FIG. 1A, but are includedin FIG. 1B for ease of comparison. Each point represents the mean±SEM.n=5 for each dose. Student's two-tailed t-test was used to compare the−LPS and the +LPS sample at each dose of ATP. FIG. 1C is a graph showingthe streptozotocin dose response. Streptozotocin was prepared as a 200mM stock in 0.1 M citrate buffer (pH 4.4) and dilutions made in mediabefore being added (10 μl) to the wells at the indicated finalconcentrations. Cells were incubated 24 h before the addition of 1.25 mMATP for 1 h and subsequent analysis. Each point represents the mean±SEM.n=5 for each dose. *p<0.05 by ANOVA followed by Tukey's post-hoc test.FIG. 1D is a graph showing the monosodiun urate dose response.Monosodiun urate crystals (InvivoGen, San Diego, Calif.) were receivedat 5 mg/ml and dilutions prepared in complete media just prior toaddition to the well (in 10 μl). Cells were incubated 24 h before theaddition of 1.25 mM ATP for 1 h and analysis. Each point represents themean±SEM. N=6 for each dose. *p<0.05 **p<0.01 by ANOVA followed byTukey's post-hoc test. FIG. 1E is a graph showing the high mobilitygroup box 1 protein (HMGB-1; ProSci, Poway, Calif.) dose response.HMGB-lwas resuspended and diluted in complete media just prior toaddition (10 μl) to the wells at the indicated final concentrations.Cells were incubated 24 h before the addition of 1.25 mM ATP for 1 h andsubsequent analysis. Each point represents the mean±SEM. n=6 for eachdose. **p<0.01 by ANOVA followed by Tukey's post-hoc test. FIG. 1F is agraph showing the N-hexanoyl-D-erythro-sphingosine dose response.N-hexanoyl-D-erythro-sphingosine (C6-Ceramide; Alfa Aesar, Haverhill,Mass.) was dissolved in DMSO and diluted in complete media prior toaddition to the wells (10 μl). Cells were incubated 24 h before theaddition of 1.25 mM ATP for 1 h and analysis. Each point represents themean±SEM. n=6 for each dose. *p<0.05 **p<0.01 by ANOVA followed byTukey's post-hoc test. FIG. 1G is a graph showing the advanced GlycationEndproduct-BSA dose response. Glycation Endproduct-BSA (AGE; Calbiochem,Millipore Sigma, Burlington, Mass.) was prepared and diluted in completemedia prior to addition to the wells. Cells were incubated 24 hourbefore the addition of 1.25 mM ATP for 1 h and analysis. Each pointrepresents the mean±SEM. n=6 for each dose. **p<0.01 ***p<0.001 by ANOVAfollowed by Tukey's post-hoc test.

FIG. 2 is a graph showing NLRP3 is activated in the urothelium duringdiabetes. Inflammasome activity (caspase-1) is increased in urotheliafrom diabetic mice as compared to non-diabetic mice. Bars are mean±SEM.n=18 (nondiabetic), 17 (diabetic). *p<0.05 by Student's two tailedt-test.

FIG. 3 are microscopy images showing that NLRP3 is present in mouseurothelia and its distribution is not effected by diabetes. All micewere examined at 15 weeks of age and all scale bars=50 μm.

FIGS. 4A-4B shows that blood glucose is not affected by knocking outNLRP3. FIG. 4A is a graph showing that blood glucose is verysignificantly increased in the diabetic mouse with a NLRP3+/+ genotype.Bars are mean±SEM. n=27 (nondiabetic), 12 (diabetic). ***p<0.0001 bypaired Student's t-test. FIG. 4B is a graph showing that blood glucoseis significantly elevated in the diabetic mouse with a NLRP3−/−genotype. Bars are mean±SEM. n=18 (nondiabetic), 21 (diabetic).***p<0.0001 by Student's two tailed t-test. ANOVA followed by Tukey'spost-hoc test was also used to compare all groups. No additionalsignificant differences were found.

FIGS. 5A-5B shows inflammation is present in the diabetic bladder and ismediated through NLRP3. FIG. 5A is a graph showing that the amount ofEvans Blue dye in the bladder was increased in diabetic mice compared tonondiabetic (both NLRP3+/+). Bars are mean±SEM. n=5 (nondiabetic), 12(diabetic). ***p<0.0001 by Student's two tailed t-test. FIG. 5B is agraph showing that diabetes did not affect the movement of Evans Blueinto the bladder in the absence of NLRP3. Bars are mean±SEM. n=6(nondiabetic), 16 (diabetic). ANOVA followed by Tukey's post-hoc testwas also used to compare all groups. No additional significantdifferences were found.

FIGS. 6A-6B show representative tracings of changes in intravesicularpressures over time from the cystometry study used to demonstrate thatNLRP3 is responsible for bladder dysfunction associated with DBD. FIG.6A is a representative tracing from the NLRP3+/+ strains. FIG. 6B is arepresentative tracings from the NLRP3−/− strains.

FIGS. 7A-71I shows that NLRP3 is responsible for bladder dysfunctionassociated with DBD. FIG. 7A is a graph showing the voiding volume innondiabetic and diabetic mice (both NLRP3+/+). FIG. 7B is a graphshowing the voiding volume in nondiabetic and diabetic mice with NLRP3deleted (NLRP3−/−). FIG. 7C is a graph showing the frequency of voidingin nondiabetic and diabetic mice (both NLRP3+/+). FIG. 7D is a graphshowing the frequency of voiding in nondiabetic and diabetic mice withNLRP3 deleted (NLRP3−/−). FIG. 7E is a graph showing the post-voidresidual (PVR) volume, or volume of urine remaining in the bladderimmediately after the last void in nondiabetic and diabetic mice (bothNLRP3+/+). FIG. 7F is a graph showing the post-void residual (PVR)volume, or volume of urine remaining in the bladder immediately afterthe last void, in nondiabetic and diabetic mice with NLRP3 deleted(NLRP3−/−). No PVR was ever detected in any of the ninenondiabetic/NLRP3+/+ mice examined. FIG. 7G is a graph showing thevoiding efficiency in nondiabetic and diabetic mice (both NLRP3+/+) ascalculated as 100(voided volume)/(voided volume+PVR). FIG. 7H is a graphshowing the voiding efficiency in nondiabetic and diabetic mice withNLRP3 deleted (NLRP3−/−) as calculated as 100(voided volume)/(voidedvolume+PVR). For all graphs, bars are mean±SEM. n=9 and 7 fornondiabetic and diabetic mice, respectively, that are NLRP3+/+. N=10 and9 for nondiabetic and diabetic mice, respectively, that are NLRP3−/−.**p<0.01, ***p<0.001 by a Student's two-tailed t-test. ANOVA followed byTukey's post-hoc test was also used to compare all groups for eachendpoint. The only additional significant differences found were in voidvolume comparing NLRP3+/+ diabetic to NLRP3−/− diabetic (p<0.05) andvoiding efficiency comparing NLRP3+/+ diabetic to NLRP3−/− nondiabetic.

FIG. 8A-8U shows NLRP3 controls changes in the densities of nervesrelated to specific DBD symptoms. FIG. 8A is a representative micrographof PGP9.5 staining (i.e. all neurons) in the bladder used to quantifynerves. The left micrograph depicts the entire transverse cross sectionstained and scanned into a TIFF file used for quantitation, as describedin the methods section. The box indicates the area zoomed in on theright micrograph to allow better visualization. Block arrow points aturothelia that stain non-specifically for PGP9.5, or at least are ofnon-neuronal origin. Arrows indicate the brown staining in the bladderwall considered to stain positive for this antigen. FIG. 8B is a graphshowing the number of PGP9.5⁺ nerves in bladder wall of 15 week micefrom nondiabetic (non diab) and diabetic (diab) mice that express NLRP3(NLRP3^(+/+)). FIG. 8C is a graph showing the same analysis as FIG. 8Bin mice that have the NLRP3 gene deleted (NLRP3). FIG. 8D is a graphshowing the size of the bladder wall in the same sections and groupsquantitated in FIG. 8B. FIG. 8E is a graph showing the size of thebladder wall in the same sections and groups quantitated in FIG. 8C.FIG. 8F is a graph showing the density of PGP9.5⁺ neurons in the samesections and groups quantitated in FIG. 8B. FIG. 8G is a graph showingdensity of PGP9.5⁺ neurons in the same sections and groups quantitatedin FIG. 8C. FIG. 8H are representative micrographs of NF-200 staining(Aδ-fibers) in the bladder used to quantify nerves. The left micrographdepicts the entire transverse cross section while the yellow boxindicates the area zoomed in on the right and arrows point at stainingin the bladder wall considered to be positive for this antigen. FIG. 8Iis a graph showing the number of Aδ-fibers in bladder wall of 15 weekmice from nondiabetic (non diab) and diabetic (diab) mice that expressNLRP3 (NLRP3^(+/+)). FIG. 8J is a graph showing the same analysis asFIG. 8I in mice that have the NLRP3 gene deleted (NLRP3^(−/−)). FIG. 8Kis a graph showing the size of the bladder wall in the same sections andgroups quantitated in FIG. 81. FIG. 8L is a graph showing the size ofthe bladder wall in the same sections and groups quantitated in FIG. 8J.FIG. 8M is a graph showing the density of PGP9.5⁺ neurons in the samesections and groups quantitated in FIG. 8I. FIG. 8N is a graph showingthe density of PGP9.5⁺ neurons in the same sections and groupsquantitated in FIG. 8J. FIG. 8O are representative micrographs of CGRPstaining (C-fibers) in the bladder used to quantify nerves. The leftmicrograph depicts the entire transverse cross section while the yellowbox indicates the area zoomed in on the right and arrows point atstaining in the bladder wall considered to be positive for this antigen.This section is also stained with the nuclear stain4′,6-diamidino-2-phenylindole (DAPI) in the coverslipping material toallow easier visualization. FIG. 8P is a graph showing the number ofC-fibers in bladder wall of 15 week mice from nondiabetic (non diab) anddiabetic (diab) mice that express NLRP3 (NLRP3^(+/+)). FIG. 8Q is agraph showing the same analysis as FIG. 8P in mice that have the NLRP3gene deleted (NLRP3^(−/−)). FIG. 8R is a graph showing the size of thebladder wall in the same sections and groups quantitated in FIG. 8P.FIG. 8S is a graph showing the size of the bladder wall in the samesections and groups quantitated in FIG. 8Q. FIG. 8T is a graph showingthe density of PGP9.5⁺ neurons in the same sections and groupsquantitated in FIG. 8P. FIG. 8U is a graph showing the density ofPGP9.5⁺ neurons in the same sections and groups quantitated in FIG. 8Q.For all graphs bars represent mean±SEM. For FIG. 8B, FIG. 8D and FIG.8F, n=11. For FIG. 8C, FIG. 8E and FIG. 8G, n=10. For FIG. 81, FIG. 8Kand FIG. 8M, n=6. For FIG. 8J, FIG. 8L and FIG. 8N, n=7. For FIG. 8P,FIG. 8R and FIG. 8T, n=4 (non diab) and 6 (diab). For FIG. 8Q, FIG. 8Sand FIG. 8U, n=3 (non diab) and 4 (diab). *P<0.05, **p<0.01, ***p<0.001by a Student's two-tailed t-test. ANOVA followed by Tukey's post-hoctest was also used to compare all groups for each endpoint. The onlyadditional significant differences found were in Aδ-fiber nerve numbercomparing NLRP3^(+/+) diabetic to NLRP3^(−/−) diabetic (p<0.05), bladderwall size in the Aδ-fiber study comparing NLRP3^(+/+) nondiabetic toNLRP3^(−/−) nondiabetic (p<0.05), Aδ-fiber nerve number comparingNLRP3^(+/+) diabetic to NLRP3^(−/−) diabetic (p<0.05). In the C-fiberstudies the NLRP3^(+/+) diabetic was significantly different from bothNLRP3^(−/−) strains in the nerve number and density graphs (p<0.05).

FIGS. 9A-9C are graphs showing that stone DAMPs activate caspase-1 in adose-dependent manner. FIG. 9A is a graph showing that CPPD activatescaspase-1 in urothelial cells in a dose-dependent manner. FIG. 9B is agraph showing that MSU activates caspase-1 in urothelial cells in adose-dependent manner. FIG. 9C is a graph showing that calcium oxalateactivates caspase-1 in urothelial cells in a dose-dependent manner. n=9for all doses of CPPD and MSU, and n=8,5,8,7,7,8,8 for the doses ofcalcium oxalate, respectively. *p<0.05; **p<0.01; ***p<0.001 by one-wayANOVA and Dunnett's post-hoc test.

FIGS. 10A-10B are graphs showing that NAC inhibits caspase-1 activationin cells treated with CPPD or MSU. FIG. 10A is a graph showing that thatNAC inhibits caspase-1 activation in urothelial cells treated with CPPD.FIG. 10B is a graph showing that that NAC inhibits caspase-1 activationin urothelial cells treated with MSU. n=9 for all NAC treatment dosesfor both CPPD and MSU. *p<0.05; **p<0.01; ***p<0.001 by one-way ANOVAand Dunnett's post-hoc test.

FIGS. 11A-11B are graphs showing that verapamil (ver) suppressescaspase-1 activation. FIG. 11A is a graph showing that ver suppressescaspase-1 activation in urothelial cells treated with CPPD. FIG. 11B isa graph showing that ver suppresses caspase-1 activation in urothelialcells treated with MSU. n=10 for all doses of Verapamil and CPPD treatedwells; n=5 for all doses of Verapamil and MSU treated wells. *p<0.05;**p<0.01; ***p<0.001 by one-way ANOVA and Dunnett's post-hoc test.

FIG. 12 is a schematic showing dosing regimen for the experiments. Ratswere given the drugs at the indicated doses at the indicated times. CP,cyclophosphamide; GLY, glyburide; FLU, fluoxetine; Veh, vehicle; Mesna,2-mercaptoethane sulfonate sodium.

FIGS. 13A-13B are graphs showing bladder weights and inflammation areincreased in response to CP and this is blocked by GLY or Mesna. FIG.13A is a graph showing that bladder weights at sacrifice were higherafter CP treatment and this was reduced by treatment with GLY or Mesna.Bars represent mean bladder weight±SEM. [Vehicle: n=32; CP: n=42; GLY:n=17; CP+GLY: n=20; CP+Mesna: n=34]. FIG. 13B is a graph showing thatinflammation in the bladder, as measured by Evans blue dyeextravasation, was increased after CP treatment and this was reduced bytreatment with GLY or Mesna. Results are reported as pg Evans bluedye/μg tissue. Bars represent mean±SEM. [Vehicle: n=3; GLY: n=3; CP:n=4; CP+GLY: n=4; CP+Mesna: n=4]. *p<0.05; **p<0.01; ***p<0.001 byone-way ANOVA and Student-Newman-Keuls post-hoc analysis.

FIGS. 14A-14B are graphs showing that Caspase-1 activity is increased inthe hippocampus, not the pons of CP-treated rats. FIG. 14A is a graphshowing hippocampus caspase-1 activity in vehicle and CP-treated rats.Results are reported as pg AFC/min/μg protein. Bars represent meanactivity±SEM. FIG. 14B is a graph showing caspase-1 activity in the ponsin vehicle and CP-treated rats. Results are reported as pg AFC/min/μgprotein. Bars represent mean activity±SEM. [For both A+B: Vehicle: n=4;CP: n=4]. *p<0.05 by two-tailed Students t-test.

FIGS. 15A-15H are graphs showing that Pro-IL-1β and Pro-IL-18 mRNAexpression levels are increased in the hippocampus during CP-inducedcystitis. NLRP3 and ASC mRNA expression levels are unchanged in thehippocampus during CP-induced cystitis. Results are expressed asrelative expression of the studied genes in treated rats compared tovehicle. Bars represent mean expression levels±SEM. FIG. 15A is a graphshowing Pro-IL-1β levels in the hippocampus [Vehicle: n=13; CP: n=12].FIG. 15B is a graph showing Pro-IL-1β levels in the pons [Vehicle: n=6;CP: n=6]. FIG. 15C is a graph showing Pro-IL-18 expression levels in thehippocampus [Vehicle: n=8; CP: n=7]. FIG. 15D is a graph showingPro-IL-18 expression levels in the Pons [Vehicle: n=4; CP n=4]. FIG. 15Eis a graph showing NLRP3 levels in the hippocampus [Vehicle: n=9; CP:n=8]. FIG. 15F is a graph showing NLRP3 levels in the pons [Vehicle:n=6; CP: n=6]. FIG. 15G is a graph showing ASC expression levels in thehippocampus [Vehicle: n=9; CP: n=8]. FIG. 15H is a graph showing ASCexpression levels in the pons [Vehicle: n=6; CP: n=6]. *p<0.05 bytwo-tailed Students t-test.

FIGS. 16A-16F show CP-induced cystitis results in inflammation andbreakdown of the blood brain barrier in the hippocampus, not in thepons. Administration of GLY or Mesna blocks this effect. FIG. 16A is agraph showing Evans blue extravasation was increased in the Hippocampusby CP. This increase was prevented by treatment with Gly or Mesna. Allresults were calculated as pg of Evans blue per μg of tissue. Barsrepresent mean±SEM. For A: Vehicle: n=3; GLY: n=3; CP: n=4; CP+GLY: n=4;CP+Mesna: n=4. For B: Vehicle: n=4; GLY: n=3; CP: n=4; CP+GLY: n=4;CP+Mesna: n=8. *p<0.05 by one-way ANOVA and Student-Newman-Keulspost-hoc analysis. FIG. 16B is a graph showing Evans blue extravasationwas not significantly changed in the pons by any treatment. FIG. 16C areimages showing CP-induced cystitis results in areas of gross blood brainbarrier breakdown, with Evans blue dye apparent (arrows) in theperiventricular region of the hippocampus. A CP-treated rat was injectedwith Evans blue as described in the Methods section. After 1 h the brainwas removed, sectioned coronally with a scalpel at the approximatelocations indicated and photographed. FIG. 16D are microscopy imagesshowing that CP results in an NLRP3-dependent increase in number ofmicroglia-like cells within the fascia dentata of the hippocampus.Coronal sections (10 μm) were cut through the hippocampus and an H&Estain was performed using routine methodical techniques. Slides werevisualized at 60×. Activated glial cells are indicated by arrows. FIG.16E are immunohistochemistry showing increased density of activatedmicroglia within the fascia dentata. Coronal sections (10 μm) were cutand Immunohistochemistry was performed using an anti-IbA1/AIF1 antibodyand routine histological methods. Slides were visualized at 20× and thenumber of microglia was quantitated. Arrows demonstrating increasedglial processes (arrows) at higher magnification are shown. FIG. 16F isa graph showing the density of Microglia. Results are depicted as thenumber of microglia per μm². Bars represent mean±SEM. [Vehicle: n=5;GLY: n=6; CP: n=7; CP+GLY: n=4; CP+Mesna: n=8]. *p<0.05 by one-way ANOVAand Student-Newman-Keuls post-hoc analysis.

FIGS. 17A-17B are graphs showing CP induces behavioral signs ofdepression through NLRP3. FIG. 17A is a graph showing the results of thesucrose preference test. A reduction of preference indicates depression.Bars represent the mean±SEM. [Vehicle: n=10; GLY: n=4; CP: n=8;CP+GLY=6; CP+Mesna=12; GP+FLU=8]. *p<0.05 and **p<0.01 by one-way ANOVAand Student-Newman-Keuls post-hoc analysis. FIG. 17B is a graph showingthe results of the forced Swim assay. An increase in time spent immobileindicates depression. Bars represent the mean±SEM. [Vehicle: n=9; GLY:n=18; CP: n=8; CP+GLY=18; CP+Mesna=6; GP+FLU=11]. *p<0.05 and **p<0.01by one-way ANOVA and Student-Newman-Keuls post-hoc analysis.

FIG. 18 is a graph showing that bladder weights are greatly increased 12weeks after BOO and this is partially inhibited by the NLRP3 inhibitorglyburide. Veh=vehicle-treated, Gly=glyburide-treated. Results are themean±SEM; ***p<0.005 by ANOVA and Student-Newman-Keuls test.(n=45,42,38,35).

FIG. 19 is a graph showing that after 12 weeks of BOO, inflammation ispresent in the hippocampus of rats. This inflammation is blocked byconcomitant treatment with glyburide. Veh=vehicle-treated,Gly=glyburide-treated. Results are the mean±SEM; *p<0.05, **p<0.01,***p<0.005 by ANOVA and Student-Newman-Keuls test. (n=8,7,12,8)

FIG. 20 is a graph showing that after 12 weeks of BOO the number ofactivated microglia in the hippocampus is increased and this increasewas blocked by glyburide treatment. The results are presented as thedensity of activated microglia per μm². Veh=vehicle-treated,Gly=glyburide-treated. Results are the mean±SEM; **p<0.01 by ANOVA andStudent-Newman-Keuls test. (n=7,6,8,7).

FIG. 21 is a graph showing that after 12 weeks of BOO, neurogenesis isstatistically decreased in the hippocampus and this increase is blockedby glyburide treatment. The results are presented as the density ofKi-67⁺ cells per μm². Veh=vehicle-treated, Gly=glyburide-treated.Results are the mean±SEM; **p<0.01 by ANOVA and Student-Newman-Keulstest. (n=6,6,6,6)

FIGS. 22A-22B are graphs showing that after 12 weeks of BOO, rats showsigns of depression. These behavior differences were not present whenrats were given glyburide or fluoxetine (Flu), an anti-depressant. FIG.22A is a graph showing that the open field assay (a measure of anxiety).The results are presented as the time in which at least 2 paws werepresent in the middle section of the open field during the 10 min testsession. Veh=vehicle-treated, Gly=glyburide-treated. Results are themean±SEM; *p<0.05, **p<0.01 by ANOVA and Student-Newman-Keuls test.(n=26,23,15,13,6). FIG. 22B is a graph showing that the sucrosepreference assay (a measure of anhedonia). The results are presented asthe amount of sucrose laden water consumed as a percentage of the totalvolume imbibed. Veh=vehicle-treated, Gly=glyburide-treated. Results arethe mean±SEM; *p<0.05, **p<0.01 by ANOVA and Student-Newman-Keuls test.(n=24,24,14,14,6).

FIGS. 23A-23B are graphs showing that after 6 weeks of BOO, inflammationis present in the hippocampus but there is no change in sucrosepreference. FIG. 23A is a graph showing Evan's blue dye extravasation isincreased in the hippocampus after 6 weeks of BOO and this increase isblocked by glyburide treatment. Following the treatments indicated, and6 weeks after BOO or sham surgery, inflammation was assessed by theEvans blue assay. Veh=vehicle-treated, Gly=glyburide-treated. Resultsare the mean±95% confidence levels; *p<0.05, **p<0.01 by ANOVA andStudent-Newman-Keuls test. (n=6,5,5,4). FIG. 23B is a graph showing thatthere is no change in sucrose preference after 6 weeks of BOO. Animalswere treated as indicated and subjected to the sucrose preference assay.Veh=vehicle-treated, Gly=glyburide-treated. Results are the mean±95%confidence levels. (n=11,8,10,8).

DETAILED DESCRIPTION OF THE DISCLOSURE

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of thedisclosure is thereby intended, such alteration and furthermodifications of the disclosure as illustrated herein, beingcontemplated as would normally occur to one skilled in the art to whichthe disclosure relates.

Definitions

Articles “a” and “an” are used herein to refer to one or to more thanone (i.e. at least one) of the grammatical object of the article. By wayof example, “an element” means at least one element and can include morethan one element.

“About” is used to provide flexibility to a numerical range endpoint byproviding that a given value may be “slightly above” or “slightly below”the endpoint without affecting the desired result.

The use herein of the terms “including,” “comprising,” or “having,” andvariations thereof, is meant to encompass the elements listed thereafterand equivalents thereof as well as additional elements. Embodimentsrecited as “including,” “comprising,” or “having” certain elements arealso contemplated as “consisting essentially of” and “consisting of”those certain elements. As used herein, “and/or” refers to andencompasses any and all possible combinations of one or more of theassociated listed items, as well as the lack of combinations whereinterpreted in the alternative (“or”).

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. Thus, the term“consisting essentially of” as used herein should not be interpreted asequivalent to “comprising.”

Moreover, the present disclosure also contemplates that in someembodiments, any feature or combination of features set forth herein canbe excluded or omitted. To illustrate, if the specification states thata complex comprises components A, B and C, it is specifically intendedthat any of A, B or C, or a combination thereof, can be omitted anddisclaimed singularly or in any combination.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise-Indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. For example, if a concentration range isstated as 1% to 50%, it is intended that values such as 2% to 40%, 10%to 30%, or 1% to 3%, etc., are expressly enumerated in thisspecification. These are only examples of what is specifically intended,and all possible combinations of numerical values between and includingthe lowest value and the highest value enumerated are to be consideredto be expressly stated in this disclosure.

As used herein, “treatment,” “therapy” and/or “therapy regimen” refer tothe clinical intervention made in response to a disease, disorder orphysiological condition manifested by a patient or to which a patientmay be susceptible. The aim of treatment includes the alleviation orprevention of symptoms, slowing or stopping the progression or worseningof a disease, disorder, or condition and/or the remission of thedisease, disorder or condition.

The term “effective amount” or “therapeutically effective amount” refersto an amount sufficient to effect beneficial or desirable biologicaland/or clinical results.

As used herein, the term “subject” and “patient” are usedinterchangeably herein and refer to both human and nonhuman animals. Theterm “nonhuman animals” of the disclosure includes all vertebrates,e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog,cat, horse, cow, chickens, amphibians, reptiles, and the like. In someembodiments, the subject comprises a human. In certain embodiments, thesubject comprises a human having a DAMP-induced or PAMP-inducedinflammation of the bladder.

“Administration” as it applies to a human, primate, mammal, mammaliansubject, animal, veterinary subject, placebo subject, research subject,experimental subject, cell, tissue, organ, or biological fluid, referswithout limitation to contact of an exogenous ligand, reagent, placebo,small molecule, pharmaceutical agent, therapeutic agent, diagnosticagent, or composition to the subject, cell, tissue, organ, or biologicalfluid, and the like. “Administration” can refer, e.g., to therapeutic,pharmacokinetic, diagnostic, research, placebo, and experimentalmethods. Treatment of a cell encompasses contact of a reagent to thecell, as well as contact of a reagent to a fluid, where the fluid is incontact with the cell. “Administration” also encompasses in vitro and exvivo treatments, e.g., of a cell, by a reagent, diagnostic, bindingcomposition, or by another cell.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs.

Inhibition of Inflammasomes for the Treatment and Prevention ofInflammation to the Bladder and Central Nervous System

The inventors have surprisingly discovered that inflammasomes may serveas therapeutic targets in the treatment of inflammation in urologicalpathologies. The inventors have discovered inflammasomes are activatedearly in the development of diabetic bladder dysfunction and cancontribute to the onset of voiding dysfunction. Furthermore, theinventors have demonstrated that NLRP3 inflammasome inhibitors canprevent or treat DBD and possibly other diabetic complications. Theinventors have also created a new murine model of diabetic mice lackingthe NLRP3 inflammasome (NLRP3^(−/−) diab).

Furthermore, the inventors have discovered that the stone componentscalcium pyrophosphate (CPPD) and monosodium urate (MSU) activate NLRP3in a reactive oxygen species (ROS) and thioredoxin-interacting protein(TXNIP)-dependent manner in bladder urothelium. These findingsdemonstrate the importance of ROS and TXNIP, and suggest that targetingeither can decrease stone-dependent NLRP3 inflammation within thebladder.

Accordingly, one aspect the present disclosure provides a method oftreating or preventing inflammation in the bladder (e.g., cystitis) in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of an inflammasome inhibitor. Inanother aspect, the present disclosure provides a method of treating orpreventing a condition that is associated with or causes inflammation inthe bladder in a subject in need thereof, comprising administering tothe subject a therapeutically effective amount of an inflammasomeinhibitor.

In another aspect, the present disclosure provides a method of treatingor preventing diabetic bladder dysfunction (DBD) in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of an inflammasome inhibitor.

Inflammation in the bladder is also known and referred to herein ascystitis. Cystitis can be acute or chronic. Acute cystitis can involvecalor, dolor, tumor, rubor (heat, pain, swelling, and redness). Chroniccan involve low-level meta-inflammation, does not typically involvecalor, dolor, tumor, or rubor, and can contribute to heart disease,cancer, diabetes, stroke, Alzheimer's disease, respiratory disease,among others. Symptoms of cystitis can include a strong, persistent urgeto urinate, a burning sensation when urinating, passing frequent, smallamounts of urine, passing cloudy urine, passing strong-smelling urine,hematuria (blood in the urine), pelvic discomfort, pressure in the lowerabdomen, and/or a low-grade fever.

Inflammation in the bladder (both acute and chronic) can be caused by avariety of different factors and conditions. Inflammation in the bladdercan be caused by, for example, bacteria (e.g., a urinary tract infectioncaused by E. coli), chemotherapy agents (e.g., cyclophosphamide andifosfamide), exposure to radiation (e.g., radiation treatment to thepelvic area), foreign-bodies (e.g., use of a catheter or a urinarystent), or chemical agents (e.g., chemicals contained in femininehygiene products, bubble bath, or other chemical that could cause anallergic reaction within the bladder).

Inflammation in the bladder can be caused by benign bladder disorders.The term benign bladder disorder refers to non-cancerous conditions thataffect the bladder. Examples of benign bladder disorders include, butare not limited to, infectious cystitis, noninfectious cystitis,reactive proliferative processes, and benign processes that secondarilyinvolve the bladder.

Inflammation in the bladder (both acute and chronic) can also be causedby an inflammatory bladder disorder. The term “inflammatory bladderdisorder” refers to a condition that can result in inflammation in thebladder. Examples of “inflammatory bladder disorders” include, but arenot limited to, diabetes, kidney stones, urinary stones, enlargedprostate, bladder outlet obstructions (BOO), interstitial cystitis (IC),benign prostatic hyperplasia (BPH), cyclophosphamide-induced hemorrhagiccystitis, diabetic uropathy (e.g., diabetic bladder dysfunction [DBD]),fibrosis, denervation, or pressure activation.

In some embodiments, inflammation in the bladder can be caused by otherdisorders and diseases including gout, benign prostatic hyperplasia(BPH), gynecological cancers (e.g., cervical cancer, ovarian cancer,uterine cancer, etc.), diabetic nephropathy, diabetic neuropathy,diabetic retinopathy, bladder cancer, pelvic inflammatory disease,endometriosis, Crohn's disease, diverticulitis, lupus, and/ortuberculosis.

Accordingly, the methods of the present disclosure provide for treatingand/or preventing conditions associated with inflammation in thebladder, including benign bladder disorders and inflammatory bladderdisorders, in a subject by administering to the subject one or moreinflammasome inhibitors.

In some embodiments, the inflammation in the bladder is caused bydiabetic uropathy. Diabetic uropathy refers to a number of debilitatingurologic complications. Types of diabetic uropathy include, but are notlimited to, DBD, urinary incontinence, urinary tract infection andsexual dysfunction.

DBD is the most common complication seen in diabetic patients. DBD is aprogressive complication. DBD can be either acute of chronic. Symptomsof acute DBD (or early stage) can include irritative voiding symptoms,including urgency (e.g., overactive bladder), frequency, nocturia,precipitancy, and urge incontinence. Symptoms of chronic (or late stage)DBD can include decompensated bladder (e.g., insensate bladder, poorcompliance, and overflow incontinence) and detrusor underactivity (DU)(also known as underactive bladder).

In some embodiments of the above aspects, the inflammation in thebladder comprises urothelial cell damage. In certain embodiments of theabove aspects, the inflammation in the bladder comprises urothelial cellinflammation. In other embodiments, bladder damage from a conditionassociated with inflammation in the bladder (e.g., diabetes) can causeneuropathy, smooth muscle dysfunction, and urothelial (barrier)dysfunction.

Inflammation in the bladder can be caused by activation of aninflammasome. In some embodiments of the above aspects, the inflammasomecan be activated by danger associated molecule patterns (DAMPs) orpathogen associated molecular patterns (PAMPs).

DAMPs are endogenous danger molecules that are released from damaged ordying cells and activate the innate immune system by interacting withpattern recognition receptors (PRRs). DAMPs can promote pathologicalinflammatory responses. DAMPs can promote the formation of a multimericstructure known as the inflammasome in macrophages and other cells,including urothelia and microglia. The molecule responsible for theDAMPs-induced inflammation in the bladder can be any molecule known todamage bladder or tissue and/or cells, including urothelial cells, orany combination thereof. Examples of DAMPs include, but are not limitedto, extracellular ATP, components of urinary stones, such as calciumpyrophosphate (CPPD), monosodium urate (MSU), and calcium oxalate, highmobility group box-1 (HMG-B1), albumin, uromodulin, uric acid crystals,hypoxia, acrolein, calcium oxalate, cholesterol, reactive oxidativespecies (ROS) serum amyloid A (SAA), amyloid β fibril, hyaluronan,aluminum, asbestos, silica, UV radiation, drusen, or skin irritants.DAMPs can also include diabetic metabolites (e.g., uric acid, glucose,MSU, HMGB1, AGE, or lipids), ROS from mitochondrial dysfunction, or K+cellular efflux.

PAMPs, on the other hand, can initiate and perpetuate the infectiouspathogen-induced inflammatory response. The pathogen responsible for thePAMPs-induced inflammation in the bladder can be any pathogen known todamage bladder tissue and/or cells, including urothelial cells, smoothmuscle cells, or any combination thereof. PAMPs can be a fungus (e.g.,Candida albicans, Saccharomyces cerevisiae, or Aspergillus fumigatus),bacteria (e.g., Listeria monocytogenes, Staphylococcus aureus,Escherichia coli, Chlamydia pneumonia, Mycobacterium tuberculosis,Clostridium difficile, Bordetella pertussis, Vibrio cholera, Neisseriagonorrhoeae, or Streptococcus pyogenes), or a virus (e.g., Influenza A,adenovirus, Sendai virus, Varicella-zoster, or herpes). In someembodiments, PAMPs can include lipopolysaccharide (LPS) from the outermembrane of the Gram-negative cell wall, bacterial flagellin, muramyldipeptide, which can be a constituent of both Gram-positive andGram-negative bacteria, alpha-hemolysin, lipoteichoic acid, or viralDNA/RNA.

As the inventors have demonstrated, inflammation in the bladder cancause secondary inflammation in the central nervous system (e.g., in thehippocampus), which can cause psychosocial maladies. In particular, theinventors have discovered a link between benign bladder disorders andmood disorders. In particular, the inventors found that cyclophosphamide(CP)-induced hemorrhagic cystitis causes NLRP3-dependent hippocampalinflammation leading to depression symptoms in rats. The inventors foundthat CP triggered an increase in inflammasome activity (caspase-1activity) in the hippocampus but not in the pons.

The inventors have also discovered that bladder outlet obstruction(BOO), a bladder-localized event, stimulates NLRP3-dependentinflammation in the rat hippocampus after 12 weeks and this inflammationcan cause depressive behavior. This is the first mechanistic explanationof the link between BOO and depression and provides evidence for adistinct bladder-brain axis.

Thus, yet another aspect of the present disclosure provides a method oftreating or preventing a condition associated with neuroinflammation,the method comprising administering a therapeutically effective amountof an inflammasome inhibitor. In some embodiments, the subject sufferingfrom neuroinflammation (or at risk for suffering from neuroinflammation)has been diagnosed with inflammation in the bladder or an inflammatorybladder disorder.

In some embodiments, a condition associated with neuroinflammation canbe a mood disorder in a subject. Mood disorders include, but are notlimited to, depression, dysthymic disorder, bipolar disorder, anxiety,anhedonia, obsessive-compulsive disorder, panic disorder, bulimia,attention deficit hyperactivity disorder (ADHD), narcolepsy, socialphobia, or post-traumatic stress disorder. In some embodiments, the mooddisorder is depression, anxiety, or anhedonia.

In other embodiments, a patient's bladder inflammation andneuroinflammation can both be treated and/or prevented concurrently byadministering an inflammasome inhibitor.

In some embodiments, the above methods further comprise administering atherapeutically effective amount of an antidepressant agent. In someembodiments, the antidepressant agent can be administered concurrentlywith, prior to, or subsequent to an inflammasome inhibitor.

The antidepressant agent can be a selective serotonin reuptakeinhibitors (SSRIs), a norepinephrine-dopamine reuptake inhibitors(NDRIs), or a monoamine oxidase inhibitors (MAOIs).

SSRIs can include, but are not limited to, citalopram, escitalopram,fluoxetine, fluvoxamine, paroxetine, and sertraline. In someembodiments, the antidepressant agent is fluoxetine.

NDRIs can include, but are not limited to, Amineptine, Bupropion,Desoxypipradrol, Dexmethylphenidate, Difemetorex, Diphenylprolinol,Ethylphenidate, Fencamfamine, Fencamine, Lefetamine,Methylenedioxypyrovalerone, Methylphenidate, Nomifensine, 0-2172,Phenylpiracetam, Pipradrol, Prolintane, Pyrovalerone, Solriamfetol,Tametraline, or WY-46824.

MAOIs can include, but are not limited to, Isocarboxazid, Nialamide,Phenelzine, Hydracarbazine, Tranylcypromine, Bifemelane, Moclobemide,Pirlindole, Toloxatone, Rasagiline, Selegiline, or Safinamide.

In certain embodiments, the subject is a mammal. In some embodiments,the mammal is a human.

Inflammasome Inhibitors

Inflammasomes are cytosolic multiprotein oligomers of the innate immunesystem responsible for the activation of inflammatory responses.Inflammasomes can include the NLR-class of inflammasomes, such as NLRP1,NLRP3, NLRP6, NLRP7, NLRP12, and NLRC4 (IPAF), as well asinterferon-inducible protein AIM2 (AIM2). The NLR-class of inflammasomeseach have a nucleotide-binding oligomerization domain (NOD), which isbound by ribonucleotide-phosphates (rNTP) and can facilitateself-oligomerization as well as a C-terminal leucine-rich repeat (LRR),which serves as a ligand-recognition domain for other receptors (e.g.TLR) or microbial ligands. The result of any inflammasome activation isthe activation of the protease caspase-1. Caspase-1 cleaves pro-IL-1βand pro-IL-18 into their active forms, which then precipitate a widerinflammatory reaction. Multiple inflammasomes are present in thebladder, including but not limited to, the NLRP1 inflammasome, the NLRP3inflammasome, the NLRP6 inflammasome, the NLRP7inflammasome, the NLRP12inflammasome, the NLRC4 inflammasome, and the AIM2 inflammasome.Multiple inflammasomes are present in the brain and spinal cord,including but not limited to, the NLRP1 inflammasome, the NLRP3inflammasome, and the NLRC4 inflammasome.

The term “NLRP1” refers to a gene that encodes NACHT, LRR, FIIND, CARDdomain and PYD domains-containing protein 1. NLRP1 can be activated byPAMPS.

The term “NLRP3” refers to NOD-like receptor family, pyrin domaincontaining 3 inflammasome or NACHT, LRR and PYD domains-containingprotein 3 (NALP3), also known as cryopyrin, cold inducedautoinflammatory syndrome 1 (CIAS1), caterpiller-like receptor 1.1(CLR1.1) or Pyrin Domain-Containing Apafl-Like Protein 1 (PYPAF1). NLRP3is a component of a multiprotein oligomer consisting of the NLRP3protein, a structural co-factor protein called thioredoxin-interactingprotein (TXNIP), ASC (apoptosis-associated speck-like protein containinga CARD) and pro-caspase 1. NLRP3 is involved in inflammation and theimmune response. In the presence of activating stimuli, this complexforms, recruits, and activates caspase-1, resulting in the cleavage andmaturation of the pro-inflammatory cytokines IL-1β and IL-18. Thesecytokines are released from the cell via a form of necrotic cell deathcalled pyroptosis, where they go on to promote inflammation.

NLRP3 can respond to both PAMPs and DAMPs.

It would be understood from context in some instances that the NLRP1inflammasome, the NLRP3 inflammasome, the NLRP6 inflammasome, theNLRP7inflammasome, the NLRP12 inflammasome, the NLRC4 inflammasome, andthe AIM2 inflammasome. Multiple inflammasomes are present in the brainand spinal cord, including but not limited to, the NLRP1 inflammasome,the NLRP3 inflammasome, and the NLRC4 inflammasome are referred toherein as NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4, and AIM2.

The term “NLRP6” refers to NOD-like receptor family pyrin domaincontaining 6, is an intracellular protein that plays a role in theimmune system. It is also known as NALP6, PYPAF5, PAN3, and CLR11.4, andis one of 14 pyrin domain containing members of the NOD-like receptorfamily of pattern recognition receptors. NLRP6 role in immunity isrelated to its ability to regulate caspase-1 and NF-κB activity.

The term “NLRP7” refers to NACHT, LRR and PYD domains-containing protein7.

The term “NLRP12” refers to NACHT, LRR and PYD domains-containingprotein 12.

The term “NLRC4” refers to NLR family CARD domain-containing protein 4.The NLRC4 protein is highly conserved across mammalian species.

The term “AIM2” refers to interferon-inducible protein AIM2.

As used herein, “inflammasome inhibitor” refers to any compound capableof inhibiting the expression and/or function of inflammasomes (e.g., theNLR3 inflammasome), in a cell, including inhibiting the expressionand/or function of the proteins in the NLRP3/IL-1β pathway. The term“inflammasome inhibitor” is meant to include one or more compoundscapable of inhibiting the expression and/or function, i.e. the term mayinclude two or more inhibitors that may be used in combination,including sequential or concomitant administration. The inflammasomeinhibitors as used with the present invention may be inflammasomespecific inhibitors. The inflammasome inhibitors may be allostericinhibitors.

Inflammasome inhibitors can include, but are not limited to, an NLRP1inflammasome inhibitor, an NLRP3 inflammasome inhibitor, an NLRP6inflammasome inhibitor, an NLRP7 inflammasome inhibitor, an NLRP12inflammasome inhibitor, an NLRC4 inflammasome inhibitor, and/or an AIM2inflammasome inhibitor.

Inflammasome inhibitors can also include compounds or a combination ofcompounds that inhibit the expression and/or function of the proteins inthe NLRP3/IL-1β pathway. Inhibitors of proteins in the NLRP3/IL-1βpathway include, but are not limited to, NLRP3 inflammasome inhibitors,TXNIP inhibitors, ASC inhibitors, NEK7 inhibitors, Gasdermin Dinhibitors, capspase-11 inhibitors, capsase-1 inhibitors, IL-1βinhibitors, IL-18 inhibitors and combinations thereof and pharmaceuticalcompositions thereof.

In some embodiments, the NLRP3 inflammasome inhibitor is a sufonylureadrug such as glyburide or functionally equivalent derivatives thereof,for example, glyburide precursors or derivatives that lack thecyclohexylurea moiety, or functionally equivalent precursors orderivatives that contain the sulfonyl and benamido groups. Examplesinclude 5-chloro-2-methoxy-N-[2-(4-sulfamoylphenyl)-ethyl]-benzamide and1-[(4-methylbenzene)sulfonyl]-1H-1,3-benzodiazol-2-amine. Functionallyequivalent precursors or derivatives of glyburide include precursors orderivatives that retain the activity of glyburide, at least in part, toinhibit or reduce the activity of NLRP3 inflammasome, e.g. that retainat least about 25% of the activity of glyburide, about 50% of theactivity of glyburide, or about 70%, 80%, or 90% of the activity ofglyburide.

In some embodiments, the NLRP3 inflammasome inhibitor is glyburide,2-mercaptoethane sulfonate sodium (Mesna), CY09, MCC950,3,4-Methylenedioxy-β-nitrostyrene (MNS), Tranilast(N-[3′,4′-dimethoxycinnamoyl]-anthranilic acid, TR), OLT1177, Oridonin,16673-34-0, JC124, FC11A-2, parthenolide, Z-VAD-FMK, Bay 11-7082, aloevera, curcumin, artesunate, dapansutrile, glybenclamide,Epigallocatechin-3-gallate (EGCG), Genipin, red ginseng extract (RGE),isoliquiritigenin (ILG), NBC 6, NBC 19 INF 39, OXSI 2, (R)-Shikonin, INF4E, CRID3 sodium salt, Mangiferin, propolis, quercetin, resveratrol, orSulforaphane (SFN), or combinations thereof.

In some embodiments, the inflammasome inhibitor is a caspase-1inhibitor. The caspase-1 inhibitor can be a direct inhibitor ofcaspase-1 enzymatic activity. Alternatively, the caspase-1 inhibitor canbe an indirect inhibitor that inhibits initiation of inflammasomeassembly or inflammasome signal propagation. Examples of caspase-1inhibitors can be antioxidants, including reactive oxygen species (ROS)inhibitors. Examples of caspase-1 inhibitors include, but are notlimited to, flavonoids including flavones such as apigenin, luteolin,and diosmin; flavonols such as myricetin, fisetin and quercetin;flavanols and polymers thereof such as catechin, gallocatechin,epicatechin, epigallocatechin, epigallocatechin-3-gallate andtheaflavin; isoflavone phytoestrogens; and stilbenoids such asresveratrol. Also included are phenolic acids and their esters such asgallic acid and salicyclic acid; terpenoids or isoprenoids such asandrographolide and parthenolide; vitamins such as vitamins A, C and E;vitamin cofactors such as co-enzyme Q10, manganese and iodide, otherorganic antioxidants such as citric acid, oxalic acid, phytic acid andalpha-lipoic acid, and Rhus verniciflua stokes extract. The caspase-1inhibitor may be a combination of these compounds, for example, acombination of a-lipoic acid, co-enzyme Q10 and vitamin E, or acombination of a caspase 1 inhibitor(s) with another inflammasomeinhibitor such as glyburide or a functionally equivalent precursor orderivative thereof.

The caspase-1 inhibitor can be a small molecule inhibitor, includingcyanopropanate-containing molecules, such as(S)-3-((S)-1-((S)-2-(4-amino-3-chlorobenzamido)-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamido)-3-cyano-propanoicacid, as well as other small molecule caspase-1 inhibitors such as(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylicacid ((2R,3 S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide. Suchinhibitors can be chemically synthesized.

In some embodiments, the caspase-1 inhibitor can be Ac-YVAD-cmk,parthenolide, INF 4E, or VX-765.

In some embodiments, the inflammasome inhibitor can be a reactive oxygenspecies (ROS) scavenger, such as N-acetylcysteine (NAC) or mannitol.

In some embodiments, the inflammasome inhibitor can be a TXNIPinhibitor, such as a calcium channel blocker (e.g., amlodipine,diltiazem, felodipine, isradipine, nicardipine, nifedipine, nisoldipine,or verapamil).

In some embodiments, the inflammasome inhibitor can be an IL-10inhibitor, such as Anakinra (Kineret), rilonacept, or canakinumab.

In some embodiments, the inflammasome inhibitor can be an ASC inhibitor,such as IC 100.

In some embodiments, the inflammasome inhibitor can be a NEK7 inhibitor,such as Oridonin (Ori).

In some embodiments, the inflammasome inhibitor can be a Gasdermin Dinhibitor, such as N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone(Ac-FLTD-CMK).

In some embodiments, the inflammasome inhibitor can be a capspase-11inhibitor. Examples of a capspase-11 inhibitor include, but are notlimited to, wedelolactone, NleF, VX-765.

Inflammasome inhibitors can be small molecules, naturally occurringmolecules (flavones, flavonoids, etc.), an interfering oligonucleotides,or an immunological inhibitor (e.g., a monoclonal antibody).

As used herein, “an interfering oligonucleotide” refers to anyoligonucleotide that interferes with, i.e. reduces, inhibits, oreliminates, the expression of an inflammasome (e.g., the NLR3inflammasome). Interfering oligonucleotides include aptamers and otheroligonucleotide molecules as described herein.

Also contemplated by the present disclosure are other types ofinhibitors of inflammasomes, including inhibitors of the NLRP3/IL-1f3pathway, including but not limited to, the following:

i. Aptamers

Aptamers, also called nucleic acid ligands, are nucleic acid moleculescharacterized by the ability to bind to a target molecule with highspecificity and high affinity. Almost every aptamer identified to dateis a non-naturally occurring molecule.

Aptamers to a given target (e.g. an inflammasome may be identifiedand/or produced by the method of Systematic Evolution of Ligands byEXponential enrichment (SELEX™) Aptamers and SELEX are described inTuerk and Gold (Systematic evolution of ligands by exponentialenrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science.1990 Aug. 3; 249(4968):505-10) and in WO91/19813.

Aptamers may be DNA or RNA molecules and may be single stranded ordouble stranded. The aptamer may comprise chemically modified nucleicacids, for example in which the sugar and/or phosphate and/or base ischemically modified. Such modifications may improve the stability of theaptamer or make the aptamer more resistant to degradation and mayinclude modification at the 2′ position of ribose.

Aptamers may be synthesized by methods which are well known to theskilled person. For example, aptamers may be chemically synthesized,e.g. on a solid support.

Solid phase synthesis may use phosphoramidite chemistry. Briefly, asolid supported nucleotide is detritylated, then coupled with a suitablyactivated nucleoside phosphoramidite to form a phosphite triesterlinkage. Capping may then occur, followed by oxidation of the phosphitetriester with an oxidant, typically iodine. The cycle may then berepeated to assemble the aptamer.

Aptamers can be thought of as the nucleic acid equivalent of monoclonalantibodies and often have K_(d)'s in the nM or pM range, e.g. less thanone of 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM. As withmonoclonal antibodies, they can be useful in virtually any situation inwhich target binding is required, including use in therapeutic anddiagnostic applications, in vitro or in vivo. In vitro diagnosticapplications can include use in detecting the presence or absence of atarget molecule.

Aptamers according to the present disclosure can be provided in purifiedor isolated form. Aptamers according to the present disclosure may beformulated as a pharmaceutical composition or medicament.

Suitable aptamers can optionally have a minimum length of one of 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides.

Suitable aptamers can optionally have a maximum length of one of 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, or 80 nucleotides.

Suitable aptamers can optionally have a length of one of 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.

ii. Oligonucleotide Repression of Inflammasome Expression

Oligonucleotide molecules, particularly RNA, can be employed to regulategene expression (e.g, expression of the NLRP1 gene, the NLRP3 gene, theNLRP6 gene, the NLRP7 gene, the NTRP12 gene, the NLRC4 gene, the AIM2gene, the ASC gene, the caspas-1 gene, and/or the TXNIP gene). Theseinclude antisense oligonucleotides, targeted degradation of mRNAs bysmall interfering RNAs (siRNAs), small molecules, post transcriptionalgene silencing (PTGs), developmentally regulated sequence-specifictranslational repression of mRNA by micro-RNAs (miRNAs) and targetedtranscriptional gene silencing.

An antisense oligonucleotide is an oligonucleotide, preferably singlestranded, that targets and binds, by complementary sequence binding, toa target oligonucleotide, e.g. mRNA. Where the target oligonucleotide isan mRNA, binding of the antisense to the mRNA blocks translation of themRNA and expression of the gene product. Antisense oligonucleotides maybe designed to bind sense genomic nucleic acid and inhibit transcriptionof a target nucleotide sequence.

In view of the known nucleic acid sequences for inflammasomes,oligonucleotides can be designed to repress or silence the expression ofinflammasome s (e.g., those regulated by the NLR-class genes). Sucholigonucleotides can have any length, but can be short, e.g. less than100 nucleotides, e.g. 10-40 nucleotides, or 20-50 nucleotides, and cancomprise a nucleotide sequence having complete- or near-complementarity(e.g. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%complementarity) to a sequence of nucleotides of corresponding length inthe target oligonucleotide, e.g. the inflammasome mRNA (e.g., the NLRP3inflammasome mRNA). The complementary region of the nucleotide sequencecan have any length, but is preferably at least 5, and optionally nomore than 50, nucleotides long, e.g. one of 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 nucleotides.

Repression of inflammasome expression (e.g., NLRP3 inflammasomeexpression) will preferably result in a decrease in the quantity ofinflammasome expressed by a cell. For example, in a given cell therepression of an inflammasome by administration of a suitable nucleicacid will result in a decrease in the quantity of inflammasome expressedby that cell relative to an untreated cell. Repression can be partial.Preferred degrees of repression are at least 50%, more preferably one ofat least 60%, 70%, 80%, 85% or 90%. A level of repression between 90%and 100% is considered a “silencing” of expression or function.

A role for the RNAi machinery and small RNAs in targeting ofheterochromatin complexes and epigenetic gene silencing at specificchromosomal loci has been demonstrated. Double-stranded RNA(dsRNA)-dependent post transcriptional silencing, also known as RNAinterference (RNAi), is a phenomenon in which dsRNA complexes can targetspecific genes of homology for silencing in a short period of time. Itacts as a signal to promote degradation of mRNA with sequence identity.A 20-nt siRNA is generally long enough to induce gene-specificsilencing, but short enough to evade host response. The decrease inexpression of targeted gene products can be extensive with 90% silencinginduced by a few molecules of siRNA. RNAi based therapeutics have beenprogressed into Phase I, II and III clinical trials for a number ofindications (Nature 2009 Jan. 22; 457(7228):426-433).

In the art, these RNA sequences are termed “short or small interferingRNAs” (siRNAs) or “microRNAs” (miRNAs) depending on their origin. Bothtypes of sequence may be used to down-regulate gene expression bybinding to complementary RNAs and either triggering mRNA elimination(RNAi) or arresting mRNA translation into protein. siRNAs are derived byprocessing of long double stranded RNAs and when found in nature aretypically of exogenous origin. Micro-interfering RNAs (miRNA) areendogenously encoded small non-coding RNAs, derived by processing ofshort hairpins. Both siRNA and miRNA can inhibit the translation ofmRNAs bearing partially complimentary target sequences without RNAcleavage and degrade mRNAs bearing fully complementary sequences.

Accordingly, the present disclosure provides the use of oligonucleotidesequences for down-regulating the expression of inflammasomes.

siRNA ligands are typically double stranded and, in order to optimizethe effectiveness of RNA mediated down-regulation of the function of atarget gene, it is preferred that the length of the siRNA molecule ischosen to ensure correct recognition of the siRNA by the RISC complexthat mediates the recognition by the siRNA of the mRNA target and sothat the siRNA is short enough to reduce a host response,

miRNA ligands are typically single stranded and have regions that arepartially complementary enabling the ligands to form a hairpin. miRNAsare RNA genes which are transcribed from DNA, but are not translatedinto protein. A DNA sequence that codes for a miRNA gene is longer thanthe miRNA. This DNA sequence includes the miRNA sequence and anapproximate reverse complement. When this DNA sequence is transcribedinto a single-stranded RNA molecule, the miRNA sequence and itsreverse-complement base pair to form a partially double stranded RNAsegment. The design of microRNA sequences is discussed in John et al,PLoS Biology, 11(2), 1862-1879, 2004.

Typically, the RNA ligands intended to mimic the effects of siRNA ormiRNA have between 10 and 40 ribonucleotides (or synthetic analoguesthereof), more preferably between 17 and 30 ribonucleotides, morepreferably between 19 and 25 ribonucleotides and most preferably between21 and 23 ribonucleotides. In some embodiments of the inventionemploying double-stranded siRNA, the molecule may have symmetric 3′overhangs, e.g. of one or two (ribo)nucleotides, typically a UU of dTdT3′ overhang. Based on the disclosure provided herein, the skilled personcan readily design suitable siRNA and miRNA sequences, for example usingresources such the Ambion siRNA finder. siRNA and miRNA sequences can besynthetically produced and added exogenously to cause genedownregulation or produced using expression systems (e.g. vectors). In apreferred embodiment the siRNA is synthesized synthetically.

Longer double stranded RNAs can be processed in the cell to producesiRNAs. The longer dsRNA molecule may have symmetric 3′ or 5′ overhangs,e.g. of one or two (ribo)nucleotides, or may have blunt ends. The longerdsRNA molecules may be 25 nucleotides or longer. Preferably, the longerdsRNA molecules are between 25 and 30 nucleotides long. More preferably,the longer dsRNA molecules are between 25 and 27 nucleotides long. Mostpreferably, the longer dsRNA molecules are 27 nucleotides in length.dsRNAs 30 nucleotides or more in length may be expressed using thevector pDECAP

Another alternative is the expression of a short hairpin RNA molecule(shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. AshRNA consists of short inverted repeats separated by a small loopsequence. One inverted repeat is complimentary to the gene target. Inthe cell the shRNA is processed by DICER into a siRNA which degrades thetarget gene mRNA and suppresses expression. The shRNA can be producedendogenously (within a cell) by transcription from a vector. shRNAs canbe produced within a cell by transfecting the cell with a vectorencoding the shRNA sequence under control of a RNA polymerase IIIpromoter such as the human H1 or 7SK promoter or a RNA polymerase IIpromoter. Alternatively, the shRNA can be synthesized exogenously (invitro) by transcription from a vector. The shRNA can then be introduceddirectly into the cell. The shRNA molecule can comprise a partialsequence of the inflammasome. The shRNA sequence can be between 40 and100 bases in length. The stem of the hairpin can be between 19 and 30base pairs in length. The stem can contain G-U pairings to stabilize thehairpin structure.

siRNA molecules, longer dsRNA molecules or miRNA molecules can be maderecombinantly by transcription of a nucleic acid sequence, preferablycontained within a vector. The siRNA molecule, longer dsRNA molecule ormiRNA molecule can comprise a partial sequence of the inflammasome.

In one embodiment, the siRNA, longer dsRNA or miRNA is producedendogenously (within a cell) by transcription from a vector. The vectorcan be introduced into the cell in any of the ways known in the art.Optionally, expression of the RNA sequence can be regulated using atissue specific (e.g. heart, liver, kidney, brain, bladder, or eyespecific) promoter. In a further embodiment, the siRNA, longer dsRNA ormiRNA is produced exogenously (in vitro) by transcription from a vector.

Suitable vectors can be oligonucleotide vectors configured to expressthe oligonucleotide agent capable of inflammasome repression. Suchvectors may be viral vectors or plasmid vectors. The therapeuticoligonucleotide may be incorporated in the genome of a viral vector andbe operably linked to a regulatory sequence, e.g. promoter, which drivesits expression. The term “operably linked” can include the situationwhere a selected nucleotide sequence and regulatory nucleotide sequenceare covalently linked in such a way as to place the expression of anucleotide sequence under the influence or control of the regulatorysequence. Thus a regulatory sequence is operably linked to a selectednucleotide sequence if the regulatory sequence is capable of effectingtranscription of a nucleotide sequence which forms part or all of theselected nucleotide sequence.

Viral vectors encoding promoter-expressed siRNA sequences are known inthe art and have the benefit of long-term expression of the therapeuticoligonucleotide. Examples include lentiviral, adenovirus, andretroviruses.

In other embodiments a vector can be configured to assist delivery ofthe therapeutic oligonucleotide to the site at which repression ofinflammasome expression is required. Such vectors typically involvecomplexing the oligonucleotide with a positively charged vector (e.g.,cationic cell penetrating peptides, cationic polymers and dendrimers,and cationic lipids); conjugating the oligonucleotide with smallmolecules (e.g., cholesterol, bile acids, and lipids), polymers,antibodies, and RNAs; or encapsulating the oligonucleotide innanoparticulate formulations.

In one embodiment, a vector can comprise a nucleic acid sequence in boththe sense and antisense orientation, such that when expressed as RNA thesense and antisense sections will associate to form a double strandedRNA.

Alternatively, siRNA molecules can be synthesized using standard solidor solution phase synthesis techniques which are known in the art.Linkages between nucleotides may be phosphodiester bonds oralternatives, for example, linking groups of the formula P(O)S,(thioate); P(S)S, (dithioate); P(O)NR′2; P(O)R′; P(O)OR6; CO; or CONR′2wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) isjoined to adjacent nucleotides through —O— or —S—.

Modified nucleotide bases can be used in addition to the naturallyoccurring bases, and may confer advantageous properties on siRNAmolecules containing them.

For example, modified bases can increase the stability of the siRNAmolecule, thereby reducing the amount required for silencing. Theprovision of modified bases can also provide siRNA molecules which aremore, or less, stable than unmodified siRNA.

The term “modified nucleotide base” encompasses nucleotides with acovalently modified base and/or sugar. For example, modified nucleotidesinclude nucleotides having sugars which are covalently attached to lowmolecular weight organic groups other than a hydroxyl group at the 3′position and other than a phosphate group at the 5′ position. Thusmodified nucleotides may also include 2′ substituted sugars such as2′-O-methyl-; 2′-O-alkyl; 2′-O-allyl; 2′-S-alkyl; 2′-S-allyl;2′-fluoro-; 2′-halo or azido-ribose, carbocyclic sugar analogues,a-anomeric sugars; epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, and sedoheptulose.

Modified nucleotides are known in the art and include alkylated purinesand pyrimidines, acylated purines and pyrimidines, and otherheterocycles. These classes of pyrimidines and purines are known in theart and include pseudoisocytosine, N4,N4-ethanocytosine,8-hydroxy-N6-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5 fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1-methyladenine,1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine,2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine,N6-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyamino methyl-2-thiouracil, -D-mannosylqueosine,5-methoxycarbonylmethyluracil, 5methoxyuracil, 2methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester,psueouracil, 2-thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil,4-thiouracil, 5methyluracil, N-uracil-5-oxyacetic acid methylester,uracil 5-oxyacetic acid, queosine, 2-thiocytosine, 5-propyluracil,5-propylcytosine, 5-ethyluracil, 5ethylcytosine, 5-butyluracil,5-pentyluracil, 5-pentylcytosine, and 2, 6,diaminopurine,methylpsuedouracil, 1-methylguanine, 1-methylcytosine.

Methods relating to the use of RNAi to silence genes in C. elegans,Drosophila, plants, and mammals are known in the art.

Accordingly, the present disclosure provides a nucleic acid that iscapable, when suitably introduced into or expressed within a mammalian,e.g. human, cell that otherwise expresses inflammasome(s) or NLRP3/IL-βpathway proteins, of suppressing inflammasome expression or expressionof NLRP3/IL-β pathway proteins by RNAi.

The nucleic acid may have substantial sequence identity to a portion ofthe inflammasome mRNA, or the complementary sequence to said mRNA.

The nucleic acid may be a double-stranded siRNA. As the skilled personwill appreciate, and as explained further below, a siRNA molecule mayinclude a short 3′ DNA sequence also.

Alternatively, the nucleic acid may be a DNA (usually double-strandedDNA) which, when transcribed in a mammalian cell, yields an RNA havingtwo complementary portions joined via a spacer, such that the RNA takesthe form of a hairpin when the complementary portions hybridize witheach other. In a mammalian cell, the hairpin structure may be cleavedfrom the molecule by the enzyme DICER, to yield two distinct, buthybridized, RNA molecules.

Only single-stranded (i.e. non self-hybridized) regions of an mRNAtranscript are expected to be suitable targets for RNAi. It is thereforeproposed that other sequences very close in the inflammasome mRNAtranscript can also be suitable targets for RNAi.

Accordingly, the present disclosure provides nucleic acids that arecapable, when suitably introduced into or expressed within a mammaliancell that otherwise expresses inflammasome(s), of suppressinginflammasome expression by RNAi, wherein the nucleic acid is generallytargeted to the sequence of, or portion thereof, of the inflammasome

By “generally targeted” the nucleic acid can target a sequence thatoverlaps with the inflammasome or is in the pathway activated by theinflammasome that causes inflammation. In particular, the nucleic acidcan target a sequence in the mRNA of a human inflammasome that isslightly longer or shorter than one of an inflammasome, but is otherwiseidentical to the native form.

It is expected that perfect identity/complementarity between the nucleicacid of the invention and the target sequence, although preferred, isnot essential. Accordingly, the nucleic acid of the invention caninclude a single mismatch compared to the mRNA of the inflammasome. Itis expected, however, that the presence of even a single mismatch islikely to lead to reduced efficiency, so the absence of mismatches ispreferred. When present, 3′ overhangs may be excluded from theconsideration of the number of mismatches.

The term “complementarity” is not limited to conventional base pairingbetween nucleic acid consisting of naturally occurring ribo- and/ordeoxyribonucleotides, but also includes base pairing between mRNA andnucleic acids of the invention that include non-natural nucleotides.

In one embodiment, the nucleic acid (herein referred to asdouble-stranded siRNA) includes the double-stranded RNA sequences forthe inflammasome. However, it is also expected that slightly shorter orlonger sequences directed to the same region of the inflammasome mRNAwill also be effective. In particular, it is expected thatdouble-stranded sequences between 17 and 23 bp in length will also beeffective.

The strands that form the double-stranded RNA may have short 3′dinucleotide overhangs, which may be DNA or RNA. The use of a 3′ DNAoverhang has no effect on siRNA activity compared to a 3′ RNA overhang,but reduces the cost of chemical synthesis of the nucleic acid strands.For this reason, DNA dinucleotides may be preferred.

When present, the dinucleotide overhangs can be symmetrical to eachother, though this is not essential. Indeed, the 3′ overhang of thesense (upper) strand is irrelevant for RNAi activity, as it does notparticipate in mRNA recognition and degradation.

Any dinucleotide overhang can therefore be used in the antisense strandof the siRNA. Nevertheless, the dinucleotide is preferably —UU or -UG(or -TT or -TG if the overhang is DNA), more preferably -UU (or -TT).The -UU (or -TT) dinucleotide overhang is most effective and isconsistent with (i.e. capable of forming part of) the RNA polymerase IIIend of transcription signal (the terminator signal is TTTTT). Thedinucleotides AA, CC and GG can also be used, but are less effective andconsequently less preferred.

Moreover, the 3′ overhangs can be omitted entirely from the siRNA.

The present disclosure also provides single-stranded nucleic acids(herein referred to as single-stranded siRNAs) respectively consistingof a component strand of one of the aforementioned double-strandednucleic acids, preferably with the 3′-overhangs, but optionally without.The present disclosure also provides kits containing pairs of suchsingle-stranded nucleic acids, which are capable of hybridizing witheach other in vitro to form the aforementioned double-stranded siRNAs,which may then be introduced into cells.

The present disclosure also provides DNA that, when transcribed in amammalian cell, yields an RNA (herein also referred to as an shRNA)having two complementary portions which are capable of self-hybridizingto produce a double-stranded motif or a sequence that differs from anyone of the aforementioned sequences by a single base pair substitution.

The complementary portions will generally be joined by a spacer, whichhas suitable length and sequence to allow the two complementary portionsto hybridize with each other. The two complementary (i.e. sense andantisense) portions may be joined 5′-3′ in either order. The spacer willtypically be a short sequence, of approximately 4-12 nucleotides,preferably 4-9 nucleotides, more preferably 6-9 nucleotides.

The 5′ end of the spacer (immediately 3′ of the upstream complementaryportion) can consist of the nucleotides -UU- or -UG-, (though the use ofthese particular dinucleotides is not essential). A suitable spacer,recommended for use in the pSuper system of OligoEngine (Seattle, Wash.,USA) is UUCAAGAGA. In this and other cases, the ends of the spacer mayhybridize with each other.

Similarly, the transcribed RNA preferably includes a 3′ overhang fromthe downstream complementary portion. Again, this can be —UU or -UG.

Such shRNA molecules may then be cleaved in the mammalian cell by theenzyme DICER to yield a double-stranded siRNA as described above, inwhich one or each strand of the hybridized dsRNA includes a 3′ overhang.

Techniques for the synthesis of the nucleic acids of the invention areof course well known in the art.

The skilled person is well able to construct suitable transcriptionvectors for the DNA of the present disclosure using well-knowntechniques and commercially available materials. In particular, the DNAwill be associated with control sequences, including a promoter and atranscription termination sequence.

Of particular suitability are the commercially available pSuper andpSuperior systems of OligoEngine (Seattle, Wash., USA). These use apolymerase-III promoter (H1) and a T₅ transcription terminator sequencethat contributes two U residues at the 3′ end of the transcript (which,after DICER processing, provide a 3′ UU overhang of one strand of thesiRNA).

The double-stranded siRNAs of the present disclosure may be introducedinto mammalian cells in vitro or in vivo using known techniques, asdescribed below, to suppress expression of the inflammasome.

Similarly, transcription vectors containing the DNAs of the presentdisclosure can be introduced into cells (e.g., bladder cells) in vitroor in vivo using known techniques, as described below, for transient orstable expression of RNA, again to suppress expression of theinflammasome.

Accordingly, the present disclosure also provides a method ofsuppressing inflammasome expression in a mammalian, e.g. human, cell,the method comprising administering to the cell a double-stranded siRNAof the present disclosure or a transcription vector of the presentdisclosure.

Similarly, the present disclosure further provides a method of treatinga pathogenically-induced and/or chemically-induced bladder inflammationor neuroinflammation in a subject in the subject, the method comprisingadministering to a subject a double-stranded siRNA of the invention or atranscription vector of the present disclosure.

The present disclosure further provides the double-stranded siRNAs ofthe present disclosure and the transcription vectors of the presentdisclosure, for use in a method of treatment, preferably a method oftreating a pathogen-induced and/or chemical-induced bladder inflammationor neuroinflammation in a subject.

The present disclosure further provides the use of the double-strandedsiRNAs of the present disclosure and the transcription vectors of thepresent disclosure in the preparation of a medicament for the treatmentof a pathogenically-induced and/or chemically-induced bladderinflammation or neuroinflammation in a subject in a subject.

The present disclosure further provides a composition comprising adouble-stranded siRNA of the present disclosure or a transcriptionvector of the present disclosure in admixture with one or morepharmaceutically acceptable carriers. Suitable carriers includelipophilic carriers or vesicles, which may assist in penetration of thecell membrane.

Materials and methods suitable for the administration of siRNA duplexesand DNA vectors of the present disclosure are well known in the art andimproved methods are under development, given the potential of RNAitechnology.

Generally, many techniques are available for introducing nucleic acidsinto mammalian cells. The choice of technique will depend on whether thenucleic acid is transferred into cultured cells in vitro or in vivo inthe cells of a patient. Techniques suitable for the transfer of nucleicacid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE dextran and calciumphosphate precipitation. In vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection.

In particular, suitable techniques for cellular administration of thenucleic acids of the present disclosure both in vitro and in vivo areknown in the art, and include: RNA interference; Gene silencing inmammals by small interfering RNAs; Gene silencing mediated by smallinterfering RNAs in mammalian cells; RNAi: gene-silencing in therapeuticintervention; Systemic delivery using liposomes: Efficient delivery ofsiRNA for inhibition of gene expression in postnatal mice; Effectiveexpression of small interfering RNA in human cells; Gene silencing bysystemic delivery of synthetic siRNAs in adult mice; Virus mediatedtransfer; Lentiviral-mediated RNA interference; Retroviral delivery ofsmall interfering RNA into primary cells; Retrovirus-delivered siRNA;Gene silencing by adenovirus-delivered siRNA; Peptide delivery. Othertechnologies that may be suitable for delivery of siRNA to the targetcells are based on nanoparticles or nanocapsules.

Another aspect of the present disclosure is a composition comprising aninflammasome inhibitor and a pharmaceutically acceptable carrier orexcipient.

In some embodiments, the inflammasome inhibitor is a NLRP3 inflammasomeinhibitor. In some embodiments, the inflammasome inhibitor is glyburide,2-mercaptoethane sulfonate sodium (Mesna), CY09, MCC950,3,4-Methylenedioxy-β-nitrostyrene (MNS), Tranilast(N-[3′,4′-dimethoxycinnamoyl]-anthranilic acid, TR), OLT1177, Oridonin,16673-34-0, JC124, FC11A-2, parthenolide, Z-VAD-FMK, Bay 11-7082, aloevera, curcumin, artesunate, dapansutrile, glybenclamide,Epigallocatechin-3-gallate (EGCG), Genipin, red ginseng extract (RGE),isoliquiritigenin (ILG), NBC 6, NBC 19 INF 39, OXSI 2, (R)-Shikonin, INF4E, CRID3 sodium salt, Mangiferin, propolis, quercetin, resveratrol, orSulforaphane (SFN), or combinations thereof.

In some embodiments, the composition comprises an inflammasome inhibitorand an antidepressant agent. In some embodiments, the antidepressantagent is fluoxetine.

Administration of Inflammasome Inhibitors

The inflammasome inhibitors may be administered to a subject, eitheralone or as a composition comprising the inflammasome inhibitor and apharmaceutically acceptable carrier/excipient (i.e., a pharmaceuticalcomposition), in an amount sufficient to induce an appropriate responsein the subject.

The present disclosure further provides for the administration to asubject an effective amount of an inflammasome inhibitor for use withthe methods disclosed herein. An “effective amount” as used herein meansan amount which provides a therapeutic or prophylactic benefit.Effective amounts of the compositions/pharmaceutical compositionsprovided herein can be determined by a physician with consideration ofindividual differences in age, weight, tumor size, extent of infectionor metastasis, and condition of the patient (subject). The optimaldosage and treatment regime for a particular patient can readily bedetermined by one skilled in the art of medicine by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

An effective amount of the composition(s) described herein can be givenin one dose, but is not restricted to one dose. Thus, the administrationcan be two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,nineteen, twenty, or more, administrations of the vaccine. Where thereis more than one administration in the present methods, theadministrations can be spaced by time intervals of one minute, twominutes, three, four, five, six, seven, eight, nine, ten, or moreminutes, by intervals of about one hour, two hours, three, four, five,six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24 hours, and so on. In the context of hours, the term“about” means plus or minus any time interval within 30 minutes. Theadministrations can also be spaced by time intervals of one day, twodays, three days, four days, five days, six days, seven days, eightdays, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days,16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinationsthereof. The invention is not limited to dosing intervals that arespaced equally in time, but encompass doses at non-equal intervals, suchas a priming schedule consisting of administration at 1 day, 4 days, 7days, and 25 days, just to provide a non-limiting example.

A “pharmaceutically acceptable excipient and/or carrier” or“diagnostically acceptable excipient and/or carrier” includes but is notlimited to, sterile distilled water, saline, phosphate bufferedsolutions, amino acid-based buffers, or bicarbonate buffered solutions.An excipient selected and the amount of excipient used will depend uponthe mode of administration. Administration comprises an injection,infusion, or a combination thereof.

An effective amount for a particular subject/patient may vary dependingon factors such as the condition being treated, the overall health ofthe patient, the route and dose of administration and the severity ofside effects. Guidance for methods of treatment and diagnosis isavailable (see, e.g., Maynard, et al. (1996) A Handbook of SOPs for GoodClinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK).

A dosing schedule of, for example, once/week, twice/week, threetimes/week, four times/week, five times/week, six times/week, seventimes/week, once every two weeks, once every three weeks, once everyfour weeks, once every five weeks, and the like, is available for theinvention. The dosing schedules encompass dosing for a total period oftime of, for example, one week, two weeks, three weeks, four weeks, fiveweeks, six weeks, two months, three months, four months, five months,six months, seven months, eight months, nine months, ten months, elevenmonths, and twelve months.

Provided are cycles of the above dosing schedules. The cycle can berepeated about, e.g., every seven days; every 14 days; every 21 days;every 28 days; every 35 days; 42 days; every 49 days; every 56 days;every 63 days; every 70 days; and the like. An interval of non-dosingcan occur between a cycle, where the interval can be about, e.g., sevendays; 14 days; 21 days; 28 days; 35 days; 42 days; 49 days; 56 days; 63days; 70 days; and the like. In this context, the term “about” meansplus or minus one day, plus or minus two days, plus or minus three days,plus or minus four days, plus or minus five days, plus or minus sixdays, or plus or minus seven days.

The composition(s) according to the present disclosure may also beadministered with one or more additional therapeutic agents. Methods forco-administration with an additional therapeutic agent are well known inthe art.

Co-administration can refer to administration at the same time in asubject, but can also include administrations that are spaced by hoursor even days, weeks, or longer, as long as the administration of the oneor more therapeutic agents is the result of a single treatment plan. Theco-administration can comprise administering the composition(s) of thepresent disclosure before, after, or at the same time as the additionaltherapeutic agent. By way of example, the composition(s) of the presentdisclosure can be given as an initial dose in a multi-day protocol, withadditional therapeutic agent(s) given on later administration days; orthe additional therapeutic agent(s) given as an initial dose in amulti-day protocol, with the composition(s) of the present disclosuregiven on later administration days. On another hand, one or moreadditional therapeutic agent(s) and the composition(s) of the presentdisclosure can be administered on alternate days in a multi-dayprotocol. In still another example, a mixture of one or more additionaltherapeutic agent(s) and the compositions of the present disclosure canbe administered concurrently. This is not meant to be a limiting list ofpossible administration protocols.

An effective amount of a therapeutic agent is one that will decrease orameliorate the symptoms normally by at least 10%, more normally by atleast 20%, most normally by at least 30%, typically by at least 40%,more typically by at least 50%, most typically by at least 60%, often byat least 70%, more often by at least 80%, and most often by at least90%, conventionally by at least 95%, more conventionally by at least99%, and most conventionally by at least 99.9%.

Formulations of the one or more therapeutic agents can be prepared forstorage by mixing with physiologically acceptable carriers, excipients,or stabilizers in the form of, e.g., lyophilized powders, slurries,aqueous solutions or suspensions.

The following Examples are provided by way of illustration and not byway of limitation.

EXAMPLES

Materials and Methods for Examples 1-7

Experimental Approach

The approach in this study is three-pronged: 1) in vitro analysis of theactivation of the inflammasome in normal mouse urothelia bydiabetic-associated DAMPS; 2) in vivo urinary function (cystometry) indiabetic mice with a genetic deletion of NLRP3 and 3) quantitation ofnerve densities in the bladders of these mice to assess potentialchanges in specific nerves thought mediate specific DBD symptoms.

Animals

All protocols adhere to the NIH Guide for the Care and Use of LaboratoryAnimals and were approved by the Institutional Animal Care and UseCommittee at Duke University Medical Center. Founder mice from TheJackson Laboratory (Bar Harbor, Mass.) consisted of Akita(C57BL/6J-Ins2Akitaa mice; stock number: 003548) (Wang et al. (1999) TheJournal of clinical investigation 103:27-37) and NLRP3^(−/−)(B6.129S6-Nlrp3^(tm1Bhk)/J (stock number: 021302) mice (Kovarova et al.(2012) Journal of immunology 189:2006-2016). While the strain of originfor the NLRP3^(−/−) mice (12956/SvEvTac) is different from the Akitabackground (C57BL/6J) these mice have been backcrossed to C57BL/6Jfor >11 generations (www.jax.org). Mice were bred by the Breeding CoreFacility at Duke University through an independently approved protocoland only female mice were used. All animals were genotyped byTransnetyx, Inc. (Cordova, Tenn.) and provided to the laboratory around4 weeks of age.

The results of genotyping were used to assign them to one of the 4experimental groups. Nondiabetics are “nondiab” and diabetics are“diab”. The groups are

-   -   1. NLRP3^(+/+), nondiab,—homozygote wt NLRP3 genes, homozygote        wt Ins2 genes—i.e. control mice    -   2. NLRP3^(+/+), diab—homozygote wt NLRP3 genes; heterozygote for        Akita mutation at the Ins2 gene—i.e. Akita diabetic control.    -   3. NLRP3^(−/−), nondiab—both NLRP3 genes knocked out, homozygote        wt Ins2 genes—i.e. NLRP3 knockout control.    -   4. NLRP3^(−/−), diab,—both NLRP3 genes knocked out, heterozygote        for Akita mutation at the Ins2 gene. This is the experimental        mouse generated for this study.

Animals were received at 5 weeks of age. Blood glucose becomes high inAkita mice (200-300 mg/dL) around 4 weeks of age and remains highthereafter (Yoshioka et al. (1997) Diabetes 1997; 46:887-894; Dolber etal. (2013) Neurourology and urodynamics 34:72-8). Mice were grown to 15weeks when DBD becomes apparent (Inouye et al. (2018) Res Rep Urol10:219-225). No changes in urinary dysfunction were found at previoustime points (Inouye et al. (2018) Res Rep Urol 10:219-225).

In Vitro Experiments

NLRP3^(+/+), nondiab mice (i.e. control littermates) were used at 7-8weeks of age. Urothelial cells were isolated (Kloskowski et al. (2014)Hum Cell 27:85-93) and plated (black-walled 96 well plates) at 50,000cells/well in 90 μl complete media [F-12K media, 10% low-endotoxindialyzed fetal bovine serum, 10 μM non-essential amino acids (allHyClone Laboratories, Logan, Utah), 1.0 μg/ml hydrocortisone(Sigma-Aldrich, St. Louis, Mo.), 10 μg/ml insulin, 5 μg/ml transferrinand 6.7 ng/ml selenium (ITS, Gibco, Gaithersburg, Md.). Following a 24hr incubation (37° C., 95% air/5% CO₂), DAMPS (10 μl) were added andincubated as indicated. 1 h prior to harvest 1.25 mM ATP was added tountreated wells. In studies that examined ATP doses, cells were platedfor 24 h, then treated with 1 μg/ml LPS (E Coli 055:B5; Sigma) in PBS orPBS alone for 24 h before treatment with the indicated doses of ATP for1 h. Caspase-1 activity was then measured as previously described(Hughes et al. (2015) Int Urol Nephrol 47:1953-1964). Controlflorescence (0 mM DAMP) was subtracted from all wells and resultsnormalized to the ATP response (except for the ATP dose responsestudies).

Histological Preparation

Bladders were formalin-fixed and paraffin-embedded in a transverseorientation. Sections (5 μm) from the lower third of the bladder werestained with anti-NLRP3 (1:100; cat #LS-C334192; Life Span BioSciences,Inc., Seattle, Wash.), anti-PGP9.5 (1:200; cat #381000; ThermoFisher,Waltham, Mass.), anti-Neurofilament 200 (NF-200; Aδ-fibers; 1:200, cat#N4142, Sigma-Aldrich, St. Louis, Mo.) or anti-Calcitonin Gene RelatedPeptide (CGRP; C-fibers; 1:80, cat #PC205L, Calbiochem, Burlington,Mass.) antibodies using standard methods and citrate antigen retrieval.Staining was visualized with secondary antibodies conjugated to eitherAlexa Fluor 488 (NLRP3, NF-200 and CGRP) or HRP (PGP9.5; developed withVectastain ABC Staining Kit; Vector Laboratories, Burlingame, Calif.).All sections were imaged on a Zeiss Axio Imager 2 microscope (Zeiss,Oberkochen, Germany) running Zen software (Zeiss). Tiling micrographsencompassing the entire cross section were captured by the software andstitched into a continuous image. Calibration bars were inserted andimages exported as TIFF files.

FAM-FLICA Caspase-1 Assay

Caspase-1 activity was assessed using the FAM-FLICA Caspase-1 Assay Kit(ImmunoChemistry Technologies, Bloomington, Minn., USA) and themanufacturer's recommended protocol. Cells were analyzed on aFACSCalibur flow cytometer (BD-Bioscience; San Jose, Calif.) (excitation488 nm, emission 533 nm) and dot plots of forward versus side scatterwere used to gate on single cells. Histograms were created and gateswere drawn to allow quantitation of the mean florescent intensity (MFI).The geometric mean of the MFI (the Geo Mean) of each sample was used forcomparisons.

Blood Glucose

Blood from the submandibular vein was assessed with the AimStrip Plusblood glucose testing system (Germaine Laboratories, San Antonio, Tex.).

Evans Blue Dye Extravasation

Extravasation of Evans blue dye is a direct measurement of vascularpermeability which is increased during inflammation. Thus, movement ofthis dye into a tissue is used as an indirect measurement ininflammation (Hughes et al. (2014) American journal of physiology Renalphysiology 306:F299-308; Hughes et al. (2016) The Journal of urology195:1598-1605; Hughes et al. (2016) J Clin Cell Immunol 7:396; Inouye etal. (2018) Res Rep Urol 10:219-225). In this study mice were injected(i.v.) with 10 mg/kg dye in saline and 1 h later sacrificed. Bladderswere weighed and incubated overnight (56° C.) in 1 ml formamide and theabsorbance (620 nM) of the formamide measured. Dye amounts werecalculated from a standard curve and normalized to bladder weight.

Cystometry

Awake restrained cystometry was performed (Hughes et al. (2014) Americanjournal of physiology Renal physiology 306:F299-308; Hughes et al.(2016) The Journal of urology 195:1598-1605; Hughes et al. (2016) J ClinCell Immunol 7:396; Inouye et al. (2018) Res Rep Urol 10:219-225). Oneweek prior, suprapubic tubes (PE-10 tubing with a flared end) wereimplanted in the bladder and secured with a purse string suture (6-0silk). The tube was externalized at the back of the neck. One week lateranimals were placed in a Ballman-type restrainer (Natsume SeisakushoCo., Tokyo, Japan) inside of a Small Animal Cystometry Lab Station (MedAssociates, St. Albans, Vt.) and positioned above an analytical balanceto measure voided volume. The catheter was connected to a syringe pumpvia an in-line pressure transducer and sterile saline infused at 15μl/min for 60-120 min. Scale and pressure readings were continuouslyrecorded with Med-CMG software (Med Associates, St. Albans, Vt.). Aftervoiding cycles stabilized (typically 3-4 cycles) an additional 3-8cycles were recorded for quantitation. Immediately after the last void,infusion was stopped, the catheter attached to a 3 ml syringe and theplunger was withdrawn for 10-15 sec to recover any PVR. CMG Analysissoftware (version 1.06; Med Associates, St. Albans, Vt.) was used toanalyze voiding cycles, defined as the time intravesicular pressurereturned to baseline after a previous void until it returned to baselinefollowing the next void. Voiding pressure is defined as the peakintravesical pressure, void volume as the amount of change on the scaleand frequency as the number of voids per hour. Voiding efficiency wascalculated as 100×the voiding volume divided by the bladder capacity(void volume+PVR).

Analysis of Nerve Densities

Quantitation of PGP9.5 and Aδ-nerve density in the bladder wall wascarried out exactly as previously described (Lutolf et al. (2018)Neurourology and urodynamics 37:952-959) while quantitation of C-fibersin the urothelium required only minor modifications. Briefly, TIFF fileswere imported into NIS-Elements software (Nikon Co., Tokyo, Japan),calibrated and the bladder wall or urothelia layer demarcated (the ROI)and area calculated. PGP9.5⁺ neurons were defined as black/brownspots>50 um². Aδ-fibers were defined as fluorescent areas>50 um² thatstained positive with a nuclear co-stain (DAPI). C-fibers were definedas continuous fluorescent fibers>1 μm. Neuronal density in a givensection was calculated by dividing the number of nerves by the μm² ofthe ROI.

Statistical Analysis

All parameters were assessed by either a two-tailed Students T-test or aone-way analysis of variance (ANOVA) followed by a Tukey's post-hocanalysis. Both analyses used GraphPad InStat software (La Jolla, Calif.)and statistical significance was defined as p<0.05.

Example 1: Diabetic DAMPS Activate the Inflammasome In Vitro

To assess the ability of diabetic DAMPS to trigger inflammasomeactivation, urothelial cells were treated in vitro and caspase-1activity measured (Hughes et al. (2015) Int Urol Nephrol 47:1953-1964).ATP was prepared as a 25 mM stock in complete media (pH adjusted with0.5 N NaOH) and dilutions made with complete media. The indicated finaldoses of ATP (in 10 μl) were then added to cells for 1 h prior tocaspase-1 analysis. ATP, the quintessential NLRP3-activating DAMP,elicited a classic dose response (FIG. 1A) and is subsequently used tocompare other DAMPs. In most cells, NLRP3 activation requires primingwith an agent such as LPS (see, e.g., Bauernfeind et al. (2013) EMBOmolecular medicine 5:814-826), However, LPS priming had no effect onthese cells (FIG. 1B). Streptozotocin poisoning of beta cells is awidely used to create a type 1 model of diabetes. However, as shown inFIG. 1C, streptozotocin directly activates the inflammasome inurothelial cells, clearly contraindicating that model for these DBDstudies. Finally, FIGS. 1D-1G demonstrates activation of caspase-1 byfour separate diabetic DAMPS (Shin et al. (2015) Ageing Res Rev24:66-76); monosodium urate (MSU), high mobility group box 1 protein(HMGB-1), C6-ceramide and advanced glycation end products (AGES).

Discussion:

The diabetic bladder is unique in that tissue damage can be caused bytwo independent mechanisms; 1) polyuria and 2) hyperglycemia. Here, thedata demonstrate that it is the NLRP3 inflammasome, located within theurothelium, senses and responds to metabolic dysregulation by initiatingan inflammatory response. Most importantly, diabetic mice lacking theNLRP3 gene do not develop diabetic bladder dysfunction.

Numerous diabetic DAMPS activated the NLRP3 inflammasome in vitro,demonstrating their pro-inflammatory potential. Interestingly,activation of NLRP3 did not require priming as in most cell types(Hughes et al. (2014) American journal of physiology Renal physiology306:F299-308). While atypical, this has been reported and suggestsurothelia either do not require priming or are already primed whenisolated, possibly through exposure to the commensal microbiome (Patelet al. (2017) Trends Mol Med 23:165-180). As shown herein,streptozotocin is an activator of NLRP3. Streptozotocin is not adiabetic metabolite but rather a pancreatic toxin commonly used toinduce diabetes in experimental models. These results discouraged theuse of that model in DBD studies.

Example 2: NLRP3 is Activated During Diabetes

To explore a role for the inflammasome in DBD it is essential todemonstrate that it is activated in the bladder by diabetes. Urotheliawere isolated and stained with a FAM-FLICA Caspase-1 Assay Kit(Immunochemistry Tech., Bloomington, Minn.) as described. All mice wereexamined at 15 weeks of age. FIG. 2 demonstrates a significant increasein active caspase-1, the enzymatic readout for active inflammasomes, inurothelia from the 15 week diabetic animals compared to nondiabeticcontrols.

Example 3: NLRP3 is Expressed in the Mouse Urothelia and itsDistribution does not Change with Diabetes

Although documented in rat (Hughes et al. (2015) Int Urol Nephrol47:1953-1964; Hughes et al. (2014) American journal of physiology Renalphysiology 306:F299-308), NLRP3 has never been examined in the mousebladder. Sections of bladder (5 μm) from the indicated mice were stainedfor NLRP3 using standard immunocytochemistry and antigen retrievalprotocols along with an Alexa Flour 488 conjugated secondary antibody.Isotype controls used normal rabbit serum instead of primary antibodies.n=3 (nondiabetic), 4 (diabetic). As shown in FIG. 3 (top left),expression of NLRP3 in the nondiabetic bladder was localized to theurothelial layer, identical to the rat. An indistinguishabledistribution was noted in the diabetic strain (FIG. 3, top right).Isotype controls showed little background staining.

Example 4: The NLRP3^(−/−) Genotype does not Affect Blood Glucose in theDiabetic

To assess a role for NLRP3 in DBD numerous endpoints were explored bothin nondiabetic and diabetic animals with intact NLRP3 (NLRP3^(+/+)) andnondiabetic and diabetic mice with NLRP3 genetically deleted(NLRP3^(−/−)). Blood glucose levels were assessed at week 15 using theAimStrip Plus blood glucose testing system. Blood glucose levels inthese groups is shown in FIGS. 4A-4B. As expected, blood glucose levelswere considerably greater in the diabetic compared to the nondiabeticmouse with NLRP3 present (NLRP3^(+/+)) (FIG. 4A). A similar increasewith diabetes is seen with the NLRP3^(−/−) strains (FIG. 4B). Nosignificant differences were detected between the nondiabetic ordiabetic based on NLRP3 expression (i.e. comparing the NLRP3^(+/+),nondiabetic to the NLRP3^(−/−), nondiabetic⁻, and likewise with thediabetics). Thus, deletion of NLRP3 has no effect on blood glucoselevels in either the nondiabetics or the diabetics.

Example 5: Inflammation is Present in the Diabetic Bladder and isMediated Through NLRP3

While there is general evidence that inflammation is present in manytissues during diabetes, there is little or no evidence in the bladder.Therefore, the Evans blue dye extravasation assay, which is a directmeasure of vascular permeability and an indirect measure ofinflammation, was used to gain insight into inflammation in the diabeticbladder. The effect of diabetes on the induction of inflammation in thebladder was assessed in the presence and absence of NLRP3 using theEvans Blue dye extravasation assay described in the Methods section. Asshown in FIG. 5A, there was a significant increase of dye extravasationin the 15-week diabetic mouse compared to the nondiabetic (bothNLRP3^(+/+)), indicating substantial inflammation at this time point.This increase in extravasation associated with diabetes was completelyblocked in the NLRP3^(−/−) mouse (FIG. 5B).

Discussion:

It has been shown that bladder inflammation and DBD develop in the Akitadiabetic mouse by 15 weeks Inouye et al. (2018) Res Rep Urol10:219-225). In that study (and this), extravasation of Evans blue waspronounced. This study also demonstrated a concurrently activation ofthe inflammasome. To investigate the role of NLRP3, the Akita mouse wascrossed with an NLRP3^(−/−) strain to create a unique substrain ofdiabetic mice lacking this inflammasome. Deletion of NLRP3 did notaffect serum glucose levels but it did abolish the inflammatory responsein the diabetic. Therefore, it appears urothelial NLRP3 is indeedcapable of sensing the metabolic dysregulation of diabetes and promotingan inflammatory response.

Example 6: NLRP3 is Responsible for Bladder Dysfunction Associated withDBD

To investigate the effects of NLRP3 on bladder dysfunction, cystometrywas performed at 15 weeks of age on the four experimental groups(Schneider et al. (2015) BJU international 115:8-15). FIGS. 6A-6B showrepresentative tracings, for each of the four groups, of the changes inpressure (cm H₂O) in the bladder lumen during several micturitioncycles, obtained during cystometry. These tracing were recorded using anin-line pressure transducer that made measurements every 0.25 s duringthe course of the experiment. The tracings were chosen to represent onlyseveral micturition cycles and do not display the entirety of therecording which was typically much longer. Voidings correspond with thelarge peaks in pressure and are indicated with asterisks (*). Thetracings from the 4 groups were arranged vertically to align the firstvoiding volume of each while continuing the recording for the samelength of time. This was to allow easy comparison and judgment ofvoiding frequency (number of voids, indicated by peaks in pressure, overtime). Tracing align the first micturition to illustrate differences inthe time between voids (the intercontraction interval) which is used tocalculate voiding frequency. Typically 3-8 micturition cycles werequantitated per animal. Not shown are tracings of the scale alignedunder the rat that measure voided volume.

Quantitative summaries are shown in FIGS. 7A-7H. The results of variousparameters measured through cystometry are shown for nondiabetic anddiabetic mice that either express NLRP3 (NLRP3+/+) or have that genedeleted (NLRP3−/−). All studies were performed at 15 weeks of age andanimals were implanted with a suprapubic catheter one week prior toanalysis. FIG. 7A demonstrates a decrease in void volume in the diabeticmice compared to the nondiabetic when NLRP3 is present (NLRP3^(+/+)).FIG. 7B shows an increase in voiding frequency in these same mice. Inanimal models, decreased void volume coupled with increased frequencycan be considered synonymous with OAB (overactive bladder), which inhumans requires subjective measures (such as urgency) that cannot bemeasured in animals. Importantly, neither of these diabetic changes wereapparent in the absence of NLRP3 (NLRP3^(−/−)) (FIG. 7B and FIG. 7D).

The development of DBD, while complex, is thought to progress from anearly, OAB phenotype to a later stage underactive bladder (UAB)characterized by increased post-void residual (PVR) volumes anddecreased voiding efficiency (Gomez et al. (2011) Current urologyreports 12:419-426). This UAB phenotype is indicative of a decompensatedbladder. In the diabetic mouse at 15 weeks (FIG. 7E and FIG. 7G) asignificant increase in PVR and decrease in voiding efficiency wasdetected, indicating the transition to UAB and decompensation had begun(FIG. 7E and FIG. 7G). However, these alterations are reduced in theNLRP3^(−/−) animals (FIG. 7F and FIG. 7H).

Discussion

Cystometrically, the diabetic animal model demonstrate clear signs ofearly DBD at 15 weeks with decreased voiding volume, increased frequencyand increased PVR. In the absence of NLRP3, diabetes did not change thefrequency or void volume, unequivocally demonstrating that NLRP3 isresponsible for the urinary changes of DBD. Interestingly, the diabeticbladder retained a significant PVR typically associated with later stageDBD and underactive bladder, thus suggesting that the transition towardsa decompensated state has begun. Importantly, in the absence of NLRP3,the bladder maintained normal voiding volumes and efficient emptying,showing the importance of NLRP3 in the transition to decompensationwhere there is a much greater risk of complications such as infectionand stone formation.

Example 7: NLRP3 Controls Changes in the Densities of Nerves Related toSpecific DBD Symptoms

DBD is associated with peripheral neuropathy and one gauge of neuropathyin a tissue is the alteration in nerve number and/or density which wouldbe expected to decrease in diabetes and may be dependent on the NLRP3inflammasome. To examine neuropathy in the bladder total nerves werequantitated using PGP9.5 as a pan neuronal marker in the bladder wall(Thompson et al. (1983) Brain research 278:224-228). Representativestaining is shown in FIG. 8A. Arrows indicate positive staining whilethe block arrow indicates nonspecific, or at least non-neuronal stainingof the urothelia. While the significance of the urothelial staining isunknown, it has been previously reported (Lutolf et al. (2018)Neurourology and urodynamics 2018; 37:952-959; Guan et al. (2015)British journal of pharmacology 172:4024-4037). Quantitatively, thetotal number of nerves in the bladder wall was decreased in the diabeticmouse (FIG. 8B) in the presence of NLRP3, but this effect was notsignificant in the NLRP3^(−/−) strain (FIG. 8C). There was no change ofbladder wall size in any group (FIG. 8D and FIG. 8E) so changes in nervedensity (FIG. 8F and FIG. 8G) directly reflect the changes in nervenumber.

Next, the specific nerve types thought to underlie individual bladdersymptoms in diabetics were assessed. First, bladder fullness is relayedto the CNS via Aδ-fibers and patients often report a reduced sensationof bladder fullness. Thus, there may be a decrease in the number and/ordensity of these fibers in the diabetic mice which may be driven by theNLRP3 inflammasome. Because Aδ-fibers are predominantly in the bladderwall, they were quantitated in this compartment. Representative stainingis shown in FIG. 811. As shown in FIG. 81, there was a significantdecrease in the number of Aδ-fibers (NF-200⁺ cells) in the bladder wallof the diabetic mouse when NLRP3 was present. This decrease was notdetected in the NLRP3^(−/−) diabetics (FIG. 8J). Bladder wall size (FIG.8K and FIG. 8L) remained constant, so changes in Aδ-fiber densities(FIG. 8M and FIG. 8N) reflect changes in fiber number.

C-fibers are associated with an OAB phenotype (38) which is common inearly stage diabetic patients and also apparent in our mice at 15 weeksof age (FIG. 7). Thus, there may be an increase in the number and/ordensity of these fibers with diabetes and this change may be driven byNLRP3. C-fibers are predominately in the urothelia and lamina propria.Representative staining is shown in FIG. 8O. As shown in FIG. 8P, thenumber of C-fibers (CGRP⁺) in the urothelium was significantly increasedin the diabetic bladders when NLRP3 was intact. This increase did notoccur in the diabetic NLRP3^(−/−) mice (FIG. 8Q). Urothelium did notchange size (FIG. 8R and FIG. 8S) so density results (FIG. 8T and FIG.8U) again reflected changes in cell numbers.

Discussion:

While traditional concepts of DBD postulated that the sole pathologicalcause was autonomic neuropathy (Kaplan et al. (1988) J DiabetComplications 2:133-139), more conventional views recognizemultifactorial disturbances (Liu et al. (2014) Chinese medical journal127:1357-1364). Considering the known association between peripheralneuropathy and the development of DBD (Tanik et al. (2016) Int NeurourolJ 2016; 20:232-23), various DBD-related symptoms could result fromdeleterious effects on the nerves within the bladder and decreased nervedensity in the bladder wall in the diabetic mice was observed.Furthermore, the effects on different types of nerves vary. TheAδ-fibers, which sense fullness in the bladder, were decreased in thebladder wall and this may explain why a diminished sense of fullness isoften reported with diabetic patients. On the other hand, the C-fiberpopulation in the urothelium increased in the diabetic bladders.C-fibers normally sense pain but they are also associated with theemergence of an overactive bladder phenotype (Fowler (2002) Urology59:37-42) which is common in early stage diabetic patients. Thus, thedifferential effects of inflammation on these two types of nervesprovide a possible explanation for the specific symptoms associated withDBD.

The current study provides a convincing mechanism whereby a plethora ofdiabetic insults converge on NLRP3 in the urothelia and translate intoinflammation and damage to the bladder. These insults include ATP andnumerous metabolites but also likely include additional insults such asreactive oxygen species, created from excessive oxidativephosphorylation, and ischemia which is a well-known activator of NLRP3that recent studies suggest play a role in DBD (Gotoh et al. (2018)Neurourology and urodynamics 37:666-672). The signal emanating from theurothelia to trigger effects in the other bladder tissues hasunidentified but likely attributable to the major products of theinflammasome, IL-1β and IL-18, acting in a paracrine fashion. Indeed ithas been shown that IL-1β is responsible for the decrease in PGP9.5⁺nerves in the bladder wall in a rat model of bladder outlet obstruction(Lutolf et al. (2018) Neurourology and urodynamics 37:952-959) and thatIL-1β is implicated it in bladder smooth muscle hypertrophy (Haldar etal. (2015) The Journal of biological chemistry 290:6574-6583).

The central role of NLRP3 in development of DBD suggests a strategy forthe prevention and management of this diabetic complication. Accordingto the DCCT trial, only 58% of patients are able to maintain the strictglycemic control favored by the American Diabetic Association (Selvin etal. (2014) Ann Intern Med 160:517-525). While strict regulation doesprevent retinopathy, nephropathy and other diabetic complications,bladder dysfunction still remains a problem for these patients (Genuthet al. (2006) Endocr Pract 12 Suppl 1:34-41; Sarma et al. (2009) Urology73:1203-1209). The present study demonstrates that NLRP3 inhibitors canprevent or treat DBD and possibly other diabetic complications wherethis pathway plays a central role.

The results clearly show that activation of the NLRP3 inflammasome,possibly by diabetic metabolites, underlies bladder dysfunction anddenervation during DBD in mice and therefore may serve as a criticalpharmacological target for combating this complication in humans.

Materials and Methods for Examples 8-10

Animals

All protocols adhere to the NIH Guide for the Care and Use of LaboratoryAnimals and were approved by the Institutional Animal Care and Usecommittee at Duke University Medical Center. Female Sprague Dawley rats(≈200 grams, 40-50 days of age, Envigo, Indianapolis, Ind.) were used inall studies.

Cell Isolation

The cell isolation protocol was modified from a previously publishedmethod (Hughes et al. (2018) Diabetes 68:430-440; Kloskowski et al.(2014) Hum Cell. 27(2):85-93). Briefly, rats were sacrificed and thebladders removed and placed in sterile PBS. Bladders were then invertedover an 18-gauge blunt tip needle, inflated with PBS, and a purse stringsuture was used to tie off the bladder. The inflated bladder was thensubmerged in Collagenase P (1 mg/ml in complete media) and shaken for 1hour at 37° C. Cells were then passed through a 40 μm nylon mesh toremove debris, pelleted and resuspended in complete media [F-12K media(HyClone Laboratories, Logan, Utah) supplemented with 10% low-endotoxindialyzed fetal bovine serum (HyClone Laboratories, Logan, Utah), 10 μMnon-essential amino acids (HyClone Laboratories, Logan, Utah), 1.0 μg/mlhydrocortisone (Sigma-Aldrich, St. Louis, Mo.), 10 μg/ml insulin (GibcoLaboratories, Gaithersburg, Md.), 5 μg/ml transferrin (GibcoLaboratories, Gaithersburg, Md.), 6.7 ng/ml selenium (GibcoLaboratories, Gaithersburg, Md.), 100 U/mL penicillin (GibcoLaboratories, Gaithersburg, Md.), and 100 μg/mL streptomycin (GibcoLaboratories, Gaithersburg, Md.)]. Cells were counted and plated at50,000 cells/well in 90 μl complete media in black-walled 96-wellplates. Cells were then incubated in a water-saturated environment for24 hours at 37° C., 95% air, and 5% CO₂. Media was removed and replacedwith fresh media (90 μL for agonist studies, 80 μL for inhibitorstudies) just prior to the start of experimental treatments.

Experimental Treatments

In vitro experiments were performed essentially as previously describedin the Materials and Methods for Examples 1-7. For agonist responsestudies, CPPD and MSU (InvivoGen, San Diego, Calif.) were prepared tothe stock concentrations of 1.25 mg/mL and 12.5 μg/mL, respectively.Using PBS (for CPPD) or complete media (for MSU), 1:2 serial dilutionswere prepared. Wells were then treated with 10 μL of the appropriatedilution of stone DAMP for a final volume of 100 μL. After treatment,cells were incubated for another 24 hours at 37° C.

For inhibition response studies, the general ROS scavengerN-acetylcysteine was prepared to the stock concentration of 5 mM for MSUtreatment and to 50 mM for CPPD treatment. Prior to treatment, the stockconcentration of NAC was buffered to pH 7.2. Verapamil was prepared to astock concentration of 1.5 mM for serial dilution and cell treatment.Using complete media for NAC and PBS for Verapamil, 1:2 serial dilutionswere prepared. Cells were treated with 10 μL of NAC or Verapamil andincubated at 37° C. for 1 or 4 hours, respectively. After pre-treatment,cells were then treated with 10 μL of CPPD (62.5 μM final) or MSU (1.25μM final) to a final volume of 100 μL. Plates were then incubated for anadditional 24 hours at 37° C. One hour prior to the end of theincubation, untreated control wells were administered 1.25 mM ATP toserve as a standard for maximal caspase response.

Caspase-1 Assay

The caspase-1 assay was performed as reported (Hughes et al. (2015) IntUrol Nephrol. 47(12):1953-1964). Briefly, media was removed and cellslysed in 50 μl lysis buffer (10 mM MgCl₂ and 0.25% Igepal CA-630) for 5minutes. An additional 50 μL of storage buffer (40 mM HEPES (pH 7.4), 20mM NaCl, 2 mM EDTA and 20% glycerol) was added, and the plates frozen at−80° C. until use (>30 minutes). Plates were then thawed and 50 μl of 50mM Hepes with 10% Sucrose and 0.1% CHAPS, 10 μl dithiothreitol (finalconcentration of 5.5 mM), and 20 μl Z-YVAD-AFC substrate (finalconcentration of 110 μM) were added to each well. Plates were thenincubated in the dark for 1 hour at 37° C. with mild shaking.Florescence was then measured (excitation 400 nm, emission 505 nm).Florescence in untreated wells (0 mM stone DAMP) was subtracted from allwells and results normalized to the ATP response. Results were reportedas a percentage of ATP response.

Statistical Analysis

Statistical analysis was performed by a one-way analysis of variancefollowed by a Dunnett's post-hoc analysis using GraphPad InStat software(La Jolla, Calif.).

Example 8: CPPD, MSU, and Calcium Oxalate Produce a Dose-DependentIncrease in Caspase-1 Activation

To determine if stone DAMPs activate caspase-1, urothelial cells wereincubated overnight prior to treatment with calcium pyrophosphate(CPPD), monosodium urate (MSU), or calcium oxalate for 24 hours.Additional wells were treated with 1.25 mM ATP for 1 hour to indicatemaximal caspase-1 activation and DAMP-treated wells were normalized tothese ATP-treated wells. CPPD triggers a robust and dose-dependentactivation of caspase-1 in isolated urothelium in vitro, with a maximalresponse of approximately 50% of the ATP response and an EC₅₀ of 62.5μg/mL (FIG. 9A). MSU was less efficacious yet more potent than CPPD,with a maximal response of 25% of the ATP response but an EC₅₀ of 0.156μg/mL (FIG. 9B). The dose response curve for CaOx is shown in FIG. 9C.

Example 9: N-Acetylcysteine Inhibits Caspase-1 Activation in CellsTreated with Stone DAMPs

To determine if CPPD and MSU signal for inflammasome activation in theurothelia through ROS, the general ROS scavenger N-acetylcysteine (NAC)was utilized. Urothelial cells were incubated overnight and then treatedwith decreasing concentrations of NAC for 1 hour before treatment with62.5 μg/mL CPPD or 1.25 μg/mL MSU for 24 hours. The caspase-1 assay wasthen performed as described in the Materials and Methods section.CPPD-treated cells had a higher IC₅₀ (625 μM) versus MSU-treated cells(IC₅₀=31.25 μM). As shown in FIG. 10A, a NAC concentration of 5 mM wassufficient to completely suppress caspase-1 activation in cells treatedwith CPPD (IC₅₀=625 μM). However, as shown in FIG. 10B, a NACconcentration of just 500 μM was sufficient to completely suppresscaspase-1 activation in MSU-treated cells (IC₅₀=31.25 04).

Discussion:

This study provides the first exploration into the urinaryDAMP-ROS-NLRP3 inflammasome pathway within the urothelium. It was foundthat NLRP3 is activated in vitro in urothelial cells by two commonstone-forming components. Importantly, it was also found that NLRP3 ismediated through an upregulation in intracellular ROS and release ofTXNIP from thioredoxin. Specifically, the general ROS scavenger NAC wasable to prevent inflammasome activation in both CPPD and MSU-treatedurothelial cells. Further, directed targeting of a ROS-responsiveprotein (TXNIP) that forms a structural component of the NLRP3inflammasome was also able to prevent inflammasome activity. Thesefindings demonstrate a ROS-driven pathway in stone-induced urothelialinflammation that relies on the TXNIP protein for functionality.

Example 10: Verapamil Inhibits Caspase-1 Activation

Verapamil (Ver) is a calcium channel blocker that has been shown todownregulate the expression of the NLRP3 binding protein TXNIP (Meloneet al. (2018) Pharm Res. 35(2):44; Xu et al. (2012) Diabetes61(4):848-856), and thus has been used in several studies to assess arole for this critical structural component of the NLRP3 inflammasome inDAMP pathways mediated by ROS (Abais et al. (2014) Journal of biologicalchemistry 289(39):27159-27168; Xu et al. (2019) Oxid Med Cell Longev1896041). Urothelial cells were incubated overnight and then treatedwith decreasing concentrations of Verapamil for 4 hours before treatmentwith 62.5 μg/mL CPPD or 1.25 μg/mL MSU for 24 hours. The caspase-1 assaywas then performed as described in the Materials and Methods section.Both CPPD and MSU-treated cells had the same IC₅₀ (100 μM). As shown inFIG. 11A, a dose of 150 μM of Verapamil was sufficient to completelyinhibit CPPD-mediated caspase-1 activation (IC₅₀=100 μM). In a similarfashion, shown in FIG. 11B, MSU-induced caspase-1 activation was alsocompletely suppressed in cells by 150 μM Verapamil (IC₅₀=100 μM).

Discussion:

Interestingly, while NAC and Verapamil both were able to abolishinflammasome activation by CPPD and MSU, there were exciting differencesin their respective abilities to do so. First, NAC doses required tocompletely inhibit inflammasome activation were higher in cells treatedwith CPPD (5 mM) compared to MSU (500 μM). This differential response ispossibly the result of differences in intracellular ROS production bythe various stone DAMPs. Second, there were no differences in treatmentdose of Verapamil required to completely inhibit inflammasome activationin either CPPD or MSU-treated cells. Maximal inhibition of caspase-1activity was seen at a Verapamil dose of 150 μM after treatment witheither stone DAMP. Therefore, it appears that functional TXNIP isrequired for the activation of NLRP3 regardless of the magnitude of ROSproduction. Based on these findings, targeted therapies aimed atimpeding different steps within this pathway may be useful to inhibitstone-mediated inflammasome activity.

NAC is an FDA-approved medication that functions as a general ROSscavenger and acts as a precursor molecule for the regeneration ofintracellular glutathione, another scavenger of ROS (Mokhtari et al.(2017) Cell J. 19(1):11-17). Clinically, NAC is very effective in anumber of patient populations, such as acetaminophen overdose,polycystic ovarian syndrome, and chronic bronchitis. While theusefulness of NAC in these patient populations is well-established, itspotential in treating urinary DAMP-mediated bladder inflammation hasnever before been explored. Our findings suggest that this drug may be auseful tool in turning off bladder inflammation caused by urinary DAMPs,thereby allowing for reduction in inflammatory complications.

Verapamil is a non-dihydropyridine calcium channel blocker used in anumber of medical conditions, such as hypertension, angina, and clusterheadaches. An early study suggested that its calcium channel blockingability might reduce the level of stone forming components in the urine,but a subsequent study did not corroborate this finding (Iguchi et al.(1993) Hinyokika Kiyo. 39(5):425-431; Sarica et al. (2007) Urologicalresearch 35(1):23-27). However, in this study, verapamil's ability todownregulate expression of TXNIP (Chen et al. (2009) American J. ofphysiology Endocrinology and metabolism. 296(5):E1133-1139; Al-Gayyar etal. (2011) British J. of pharmacology 164(1):170-180) proved a usefultool to block stone-DAMP activation of NLRP3. Therefore, while it maynot affect the concentration of stone forming moieties, it may actuallyprotect the urinary tract from these pro-inflammatory urinarycomponents.

This study demonstrates that urinary stone-forming DAMPS activate NLRP3in urothelial cells via ROS production and require the presence ofTXNIP. This pathway is effectively blocked by the administration of theanti-oxidant, NAC, or by down-regulating TXNIP expression withverapamil. These agents can be useful in preventing lower urinary tractinflammation, pain, and risk of fibrosis and scarring in stone formingpatients. More broadly, and the subject of future studies, many otherurinary DAMPs could also activate the NLRP3 inflammasome and provoke anurothelial inflammatory response via the same pathway demonstrated inthis investigation. It is well-known that consumption of certain foods,such as chocolate, alcohol, acidic juices, can exacerbate lower urinarytract symptoms in sensitive patients. Abstinence from these dietaryfactors is very effective, but that requires the identification of thespecific item which can be challenging. If the pathway elucidated hereis common to many other urinary DAMPs, then targeting ROS production,TXNIP expression and/or NLRP3 activation could be implemented as atreatment strategy even if the inciting factor is unknown. Additionally,it has been shown in Example 1 that diabetic metabolites are capable ofactivating NLRP3 in urothelium and this contributes to the developmentof diabetic bladder dysfunction (DBD). If future studies demonstratethat these metabolites have the same mechanism of action as the stoneDAMPs (uric acid being an example of both) then targeting ROS productionor TXNIP expression may be useful in preventing DBD.

Conclusion

Urinary stones are comprised of components that induce a state ofinflammation, which can affect patients in a number of clinicallymeaningful ways. The present study suggests that this inflammation isdue, in part, to the activation of the NLRP3 inflammasome by increasedintracellular concentrations of ROS that impinge upon TXNIP.Importantly, this study illuminates steps in the pathway that can bepharmacologically targeted to potentially reduce complications ofinflammation in stone patients.

Materials and Methods for Examples 11-15

Animals

All protocols adhere to the NIH Guide for the Care and Use of Laboratoryanimals and were approved by the Institutional Animal Care and UseCommittee at Duke University Medical Center. Female Sprague Dawley Rats(˜200 g) were randomly divided into groups to receive the varioustreatments shown in Table 1.

TABLE 1 Groups and Treatments Group Vehicle Gly CP Mesna FluoxetineControl 10% EtOH Glyburide 2.5 mg/kg CP 150 mg/kg CP + Gly 2.5 mg/kg 150mg/kg CP + Mesna 150 mg/kg 40 mg/kg CP fluoxetine 5 mg/kg

Not all treatments groups were used for all end points. The rats werethen subjected to the dosing regimen shown in FIG. 12. Basically,animals were injected i.p. with a single dose of CP (150 mg/kg) or PBSas a control and 24 hr later end point analysis began. However,depending on the experiment, rats were also pretreated/treated withvarious inhibitors to assess the role of various pathways. To assess arole for NLRP3, rats were given the NLRP3 inhibitor glyburide (Lamkanfiet al. (2009) The Journal of cell biology 187: 61-70) (GLY; 2.5 mg/kg in10% ethanol in PBS, ≈800 μl/rat, p.o., (Hughes et al. (2014) Americanjournal of physiology Renal physiology 306: F299-308) or vehicle as thecontrol. GLY was given in the evening prior to CP administration (5 PM)and again 16 h later (9 AM). Four h later (1 PM), CP or PBS alone wasinjected (i.p). Additional doses of GLY were given 4 h (5 PM) and 20 h(9 AM) after CP injection. 24 h after CP injection animals weresacrificed or entered into the Evans blue protocol or behavior assays.To assess a role for acrolein-induced cystitis, Mesna (40 mg/kg, (Ali etal. (2014) Indian J Pharmacol 46: 105-108) was administered to a groupof animals 4 h before CP, immediately before CP, 4 h and 16 h after CP.For behavior assays, an additional group of rats was administered theantidepressant fluoxetine (FLU) as a control. FLU was given 48 h, 24 hand 4 h prior to CP administration (and 20 h after). This proved tominimum time necessary to see effects of fluoxetine, which are mostnoted chronically but acute effects have been reported (Silva et al.(1999) Braz J Med Biol Res 32: 333-339).

Bladder Weight

Bladder weights were recorded at the time of euthanasia.

Evans Blue Assay and Gross Analysis

Inflammation in the bladder and inflammation/blood brain barrierpermeability in the brain was measured using Evans blue dye (Belayev etal. (1996) Brain research 739: 88-96; Michels et al. (2015) Brain BehavImmun 43: 54-59). Rats were injected i.v. in the tail vein (2%, 3ml/kg). One hour after injection, rats were euthanized andtranscardially perfused through a ventricular catheter to removeintravascular dye. For gross analysis brains were isolated and sectionedcoronally using a scalpel and photographed. For other analyses bladderswere removed, weighed and placed into 1 ml formamide. The hippocampusand pons were dissected out, weighed and placed into 250 μl formamide.Samples were then incubated at 56° C. overnight with shaking. Absorbancewas measured (620 nm) and the results calculated as pg Evans blue/μgtissue using a standard curve of Evans blue absorbance.

Caspase-1 Activity

Caspase-1 activity was measured using a fluorometric assay as previouslydescribed (28). Briefly, samples were homogenized in 200 μl of LysisBuffer (10 mM MgCl₂, 0.25% Igepal CA-630), centrifuged (10,000×g, 10min) and the supernatant mixed with equal parts Storage Buffer [40 mMHepes (pH 7.4), 20 mM NaCl, 2 mM EDTA, 20% glycerol. Extract (75 μl) wascombined with 25 μl of a 1:1 combination of Lysis and Storage buffer, 50μl assay buffer [25 mm HEPES (pH 7.5), 5% sucrose, 0.05% CHAPS], 10 μl100 mM DTT and 20 μl 1 mMN-acetyl-Tyr-Val-Ala-Asp-7-Amino-4-trifluoromethylcoumarin (Ac-YVAD-AFC)in blacked-walled 96-well plates. Plates were incubated at 37° C. for 1hour in the dark with mild shaking. Fluorescence (excitation: 400 nm,emission: 505 nm) was measured and compared with a standard curve offluorescence versus free AFC to determine the rate of productproduction. Protein concentrations of sample aliquots were assessed byBradford assay (Bradford (2004) Anal Biochem 72: 248-254) and rates werenormalized to protein to calculate the specific activity of caspase-1.

Quantitative PCR (qPCR)

qPCR was performed by Gene Master LLC (Cary, N.C.) using their standardtechniques. RNA was extracted using Trizol (Thermo Fisher, Waltham,Mass.) according to manufacturer's protocol and samples stored at −20°C. until transferred to Gene Master (<2 weeks). cDNA was then generated(Invitrogen Superscript III kit) and qPCR run in triplicate withvalidated primers (pro-IL-1β: forward primer: caccttcttttccttcatctttg(SEQ ID NO:01), reverse primer: tcgttgcttgtctctccttg (SEQ ID NO:02);pro-IL-18: forward primer: aggctcttgtgtcaacttcaaa (SEQ ID NO:03),reverse primer: agtctggtctgggattcgtt (SEQ ID NO:04); NLRP3: forwardprimer: gaagattacccacccgagaaa (SEQ ID NO:05), reverse primer:ccagcaaacctatccactcc (SEQ ID NO:06); ASC: forward primer:atctggaggggtatggcttg (SEQ ID NO:07), reverse primer:cttgttttggttgggggtct (SEQ ID NO:08)) using β-actin (forward primer:cccattgaacacggcatt (SEQ ID NO:09), reverse primer: accagaggcatacagggaca(SEQ ID NO:10)) as an internal control. Gene expression levels ofvehicle-treated rats were averaged and normalized to a value of one.Results are presented as relative expression (fold increase) of thestudied genes in treated rats compared to vehicle.

Histological Analysis

Whole brains were immersed in 10% neutral buffered formalin (RT, 48 h),sliced coronally and embedded in paraffin blocks with the cuthippocampal plane on the block face. Sections (10 μm) were then cut andstained with hematoxylin and eosin using routine methods. Sections werevisualized using Olympus Vanox BH-2 microscope and analyzed by aboard-certified pathologist (WTH) for evidence of inflammation andchanges in microglia.

Immunocytochemistry and Quantitation of Microglia

Coronal sections (10 μm) of the hippocampus were stained withanti-IbA1/AIF1 (1:500) (catalog NBP2-19019; Novus Biologicals,Centennial, Colo.) using standard methods and citrate antigen retrieval.HRP development was accomplished with the Vectastain ABC Staining Kit(Vector Laboratories, Burlingame, Calif.) using a secondary antibodyprovided. All sections were imaged on a Zeiss Axio Imager 2 microscope(Zeiss, Oberkochen, Germany) running Zen software (Zeiss), using thetiling and stitching feature to ensure the entire fascia dentata wasvisualized. Images were imported into NIS-Elements software (Nikon Co.,Tokyo, Japan). One hemisphere was chosen and 600,000-700,000 μm² of thefascia dentata was demarcated as the region of interest (ROI). Thenumber of microglia within this region was then counted. Microglia cellswere defined as black/brown spots with two or greater associatedtendrils and the number present in the ROI was counted. Microglialdensity was then calculated.

Behavioral Assays

Depressive symptoms were measured using the sucrose preference assay andthe forced swim assay. These assays began 24 h after CP treatment andrequired 24-48 h to complete. No additional medications were given tothe animals during this period. In the sucrose preference assay, animalswere presented two bottles simultaneously, one containing a 2% sucrosesolution and the other containing drinking water. The amount of liquidconsumed in each bottle during the 24 h of testing was measured. Thelocation of the two bottles was varied during this period. The sucrosepreference score was expressed as percent of total liquid intake.

In the forced swim assay, rats were placed in tap water (25-27° C.)within a large, clear cylinder (Harvard Apparatus; Holliston, Mass. Cat#76-0494), such that the animal's legs nor their tail touched thebottom. Rats were left in this cylinder for a 10 min training period. 24h later (48 h after CP), rats were then placed back in the cylinder andrecorded for 5 min (Logitech Webcam; Silicon Valley, Calif.). Therecordings were quantified for time spent immobile by several blindedindividuals and the average score used in each individual measurement.Immobility was defined as absence of movement except for those necessaryfor keeping the nose above water.

Statistical Analysis

Statistical differences were assessed using a two-tail Students t-testor ANOVA followed by a Student-Newman-Keuls post-hoc analysis, asindicated in the figure legends. All statistical analyses were conductedusing Graph Pad In Stat Software (La Jolla, Calif.) and results wereconsidered significant if p<0.05.

Example 11: CP Administration Increased Bladder Weight and Inflammation

Bladder weight and inflammation was used to confirm effective inductionof cystitis. As shown in FIG. 13A, rat bladder weights weresignificantly increased 24 h after CP administration. Additionally,there was a significant increase in inflammation as indicated byextravasation of Evans blue (FIG. 13B). Both bladder weight andinflammation were reduced when CP-treated rats also received GLY,although the levels were still higher than vehicle-treated or GLY-onlytreated rats (FIG. 13A and FIG. 13B). As expected, concomitant treatmentwith Mesna blocked the changes in both endpoints to levels notsignificantly different from vehicle-treated controls (FIG. 13A and FIG.13B).

Example 12: Caspase-1 Activity is Increased in the Hippocampus, but notin the Pons

CNS tissue was harvested and processed as described in the Materials andMethods section. As shown in FIG. 14A, there was a significant increasein caspase-1 activity, a marker of inflammasome activation, in thehippocampus of CP-treated rats at 24 h. This increase was not present inthe pons (FIG. 14B). This suggests that central activation of theinflammasome in response to CP-induced cystitis is occurring, at leastin part, within the hippocampus and that this effect is specific and nota general response in the brain to CP or its metabolites.

Discussion:

Chronic inflammatory syndromes are present in every specialty inmedicine. Whether it is irritable bowel syndrome in gastroenterology orinterstitial cystitis in urology, these conditions present a myriad ofchallenges to physicians and patients. These patients have high rates ofco-morbid depression, anxiety and other related psychiatric disordersand recent studies have begun to demonstrate this adverse psychosocialconsequence may be due to neuroinflammation mediated by the NLRP3inflammasome (Miller et al. (2016) Nature reviews Immunology 16: 22-34).The present study has shown for the first time that an acute insult tothe bladder in an animal model can also result in neuroinflammation inthe CNS and the associated symptoms of depression,

The studies began by looking for signs of inflammasome activation(caspase-1 activity) in the hippocampus, due to its known associationwith depression, and the pons, due to its well-known function inmicturition. An increase within the hippocampus 24 h after CP-treatmentwas found, but there were no significant changes in the pons. Thisdifferential response suggests the effect is specific to this region andnot a non-specific, perhaps toxic, response of the brain to CP or itsmetabolites.

Example 13: Pro-IL-1β and Pro-IL-18 mRNA Expression are Increased in theHippocampus

Gene expression of pro-IL-1β and pro-IL-18 was measured in thehippocampus and pons. FIG. 15A demonstrates a significant increase inpro-IL-1β expression in the hippocampus of CP-treated rats while nochange in expression was found within the pons (FIG. 15B). A significantincrease of pro-IL-18 gene expression was also found in the hippocampusof CP-treated rats (FIG. 15C). Interestingly, there was also an increasein pro-IL-18 expression in the pons of CP-treated rats (FIG. 15D).

NLRP3, and other critical components of the inflammasome such as theassociated speck-like protein containing a COOH-terminal caspaserecruitment domain (ASC), have been found to be upregulated in manyother inflammatory conditions, although their expression is regulated bymechanisms different than those regulating pro-IL-1β and pro-IL-18 (54).However, as shown in in FIG. 15E-FIG. 1511, no significant changes wereobserved in either NLRP3 or ASC in the hippocampus or pons of CP-treatedrats.

Discussion:

Often associated with inflammasome activation is an increase inexpression of pro-IL-1β and/or pro-IL-18. Increased mRNA expression ofboth of these pro-inflammatory cytokines in the hippocampus wasobserved, consistent with inflammasome activation or at least thebeginning of an inflammatory response. Surprisingly, pro-IL-18 wassignificantly increased in the pons suggesting this region of the brainis actually responding to CP. However, no other indication of aninflammatory reaction was observed.

Example 14: CP-Induced Cystitis Induces NLRP3-Dependent Inflammation inthe Hippocampus

Breakdown of the blood brain barrier is one of the known consequences ofNLRP3-induced inflammation within the central nervous system (Song etal. (2017) Front Cell Neurosci 11: 63). In order to evaluate thischange, the Evans blue assay was performed. In the hippocampus fromCP-treated rats, a significant increase in Evans blue extravasationcompared to vehicle-treated or GLY only-treated controls was detected,indicating inflammation and disruption of the blood brain barrier (FIG.16A). Critically, the administration of either GLY or Mesna at the timesindicated in FIG. 12 reduced the dye extravasation to levels notsignificantly different from controls. In the pons (FIG. 16B) there wasno significant change in dye extravasation in response to CP, clearlydemonstrating the effect in the hippocampus was specific and not ageneral breakdown of this barrier. When gross cross sections of thebrain from CP-treated rats were examined (FIG. 16C), areas of Evans bluedye were apparent in the periventricular region of the hippocampalformation, where the dye was permeating through areas of trueblood-brain barrier breakdown and not through the circumventricularorgans. These blue areas were not observed in any other group (data notshown).

Histologically, the hippocampus demonstrated evidence of inflammation inthe CP-treated rats (FIG. 16D). In particular, cells with morphology ofactivated microglia were present in the CP-treated samples (arrows inupper right and lower right panels). These inflammatory changes werefound predominantly in and around the fascia dentata (indicated bybrackets in the upper left panel). The activated microglia were notpresent when the CP-treated rats were administered GLY (CP+GLY groupshown in lower left panel, all other groups—data not shown). Toquantitate these changes, hippocampal sections were stained forIbA1/AIF1, a marker of activated microglia, and the density of activatedmicroglia in the fascia dentata region quantitated. FIG. 16E shows atypical staining pattern for control, CP and CP+GLY samples (othergroups not shown). FIG. 16F shows the results of this quantitation witha significantly increased density of microglia in the CP-treated rat.This increase was blocked to levels not significantly different fromcontrols when rats were treated with either GLY or Mesna. Qualitatively,we also noted an increase in microglial processes in brains fromCP-treated rats (arrows in FIG. 15E lower right panel).

Discussion:

One of the most significant changes discovered was that CP-inducedcystitis triggers breakdown of the blood-brain barrier within thehippocampus, but not the pons. The demarcation between the hippocampusand pons confirms that neither CP itself nor its metabolites aredirectly causing this breakdown. If direct effects were involved, thebreakdown could be expected to occur throughout the CNS and not localizeto the hippocampus. It should be noted that these experiments do notrule out inflammation and inflammasome activation in other parts of thebrain. Importantly, glyburide prevented this blood brain barrierbreakdown. Seeing as glyburide is an inhibitor of NLRP3 with little orno effects on other inflammasomes (Lamkanfi et al. (2009) The Journal ofcell biology 187: 61-70), these results specifically implicate the NLRP3inflammasome in this response. Finally, the breakdown of this barrierwas also blocked with Mesna which is well-known to bind acrolein in theurine and prevent it from harming urothelia. Mesna undergoes rapidoxidation in plasma with only a very small portion remaining incirculation. Thus, urinary Mesna concentration vastly exceed that inplasma, essentially restricting this compound to the urinary system(Carless et al. (2008) 17th Expert Committee on the Selection and Use ofEssential Medicines; Jenkins (2014) London Cancer). Indeed, doses up to100 mg/kg (compare to 40 mg/kg used in this study) have produced noapparent effects on bone marrow, hepatic, renal or CNS function (Id.).Thus, Mesna's effectiveness in this study strongly argues that thebreakdown of the blood brain barrier in the hippocampus is a directresult of the cystitis triggered by CP in the bladder.

While Evan's blue does detect breakdown of the blood brain barrier it isalso a harbinger of inflammation in a tissue. Indeed, histologicalevidence of inflammation was found in the CP-treated rats within thefascia dentata. Quantitation of the activated microglia in this regionclearly showed an increased density and this increase was again blockedby both glyburide and Mesna. Thus, these results demonstrate that theNLRP3 inflammasome plays a critical role in inducing inflammation in thehippocampus in response to CP-induced cystitis. Therapeutically,administration of an NLRP3 inhibitor at the time of an acuteinflammatory event, can serve as a critical intervention to prevent theemergence of neuroinflammatory changes.

At this time, the mechanism by which inflammation in the bladder resultsin inflammation in the hippocampus is unclear. Currently there are 3distinct pathways that may contribute to varying degrees (Miller et al.(2016) Nature reviews Immunology). The first pathway, called the humoralpathway, includes the leaking of peripheral cytokines directly into theCNS through areas of blood brain barrier breakdown. Given serumpro-inflammatory cytokines (such as IL-1β, TNF-α, and IL-6) areincreased in response to CP (Kim et al. (2015) Biomol Ther (Seoul) 23:180-188), and given that barrier breakdown was detected as describedherein, this pathway likely contributes to CP-induction ofneuroinflammation. Peripheral cytokines may also be directly transportedacross the barrier through saturable transport molecules (Miller et al.(2016) Nature reviews Immunology 16: 22-34). The second pathway, theneural pathway, involves cytokine stimulation of afferent nerves thatcarry retrograde transmission of a signal for inflammation throughascending fibers and into the brain where they are translated back intocentral cytokine signals (Miller et al. (2009) Biol Psychiatry 65:732-741). Recent evidence has established an inflammatory phenotypewithin the L6-S1 dorsal quadrants of the spinal cord in CP-cystitis (Liuet al. (2016) Mol Pain 12), suggesting that neural transfer of aninflammatory response may also be playing a role in moving the signalfrom the bladder to the CNS. Likewise, CP-cystitis is well known tocause bladder pain and acute pain itself can directly stimulate symptomsof depression (Michaelides et al. (2019) Postgrad Med 131: 438-444),most likely through this neural transfer pathway. Finally, a thirdpathway, called the cellular pathway, has been found to contribute tothe transfer of inflammatory signals (D'Mello et al. (2009) J Neurosci29: 2089-2102). This pathway involves movement of immune cells that havebeen activated in the periphery directly to the brain vasculature andparenchyma. This pathway was discovered in studies of the inflamedliver, which releases TNF-α. TNF-α crosses the blood brain barrier andstimulates the release of CC-chemokine ligand 2 (CCL2) from microgliawhich, in turn, passes back into the periphery and triggers thechemotaxis of monocytes into the brain (Id.). As stated earlier, thereare increases in serum levels of TNF-α in response to CP (Kim et al(2015) Biomol Ther (Seoul) 23: 180-188), so it is possible this pathwayis involved as well.

Example 15: CP-Induced Cystitis Results in NLRP3-Dependent Symptoms ofDepression

To determine if CP-induced cystitis results in depressive symptoms, twoindependent behavioral assays were performed, the sucrose preferenceassay and the forced swim assay. For these studies, an additionalcontrol group of CP-treated rats were administered the antidepressantfluoxetine to differentiate true depression symptoms from sick behavior,which would not be effected by the antidepressant. In the sucrosepreference assay (FIG. 17A), CP-treated rats consumed a significantlylower percentage of sucrose-laden water. Importantly, this change wasprevented when CP-treated rats were treated with GLY, Mesna orfluoxetine. In the forced swim assay (FIG. 17B), CP-treated rats spentsignificantly more time immobile when compared to control. Critically,this change was also prevented by GLY, Mesna and fluoxetine.

Discussion:

Regardless of how the peripheral inflammatory signal is transferred,once the brain is inflamed it is well known to bring about symptoms ofdepression and other negative psychosocial behaviors (Miller et al.(2009) Biol Psychiatry 65:732-741; Miller et al. (2016) Nature reviewsImmunology 16:22-34; Noto et al. (2014) Neuroimmunomodulation21:131-139; Sayana et al. (2017) J Psychiatr Res 92:160-182; Teixeira etal. (2014) Neuroimmunomodulation 21: 71). Indeed, using two distinct andwell-established assays of depression, it was found that symptoms ofdepression strongly correlated with neuroinflammation in the presentstudy. While these studies do not address how neuroinflammation actuallybrings about these symptoms, numerous theories abound in the literature(Miller et al. (2009) Biol Psychiatry 65:732-741; Miller et al. (2016)Nature reviews Immunology 16:22-34) suggesting the mechanism may bemultifactorial. For example, cytokines signals are known to influencethe availability of mood-relevant neurotransmitters, particularly themonoamines (Miller (2009) Brain Behav Immun 23: 149-158). Many of thesesignals, working through well-known STAT, IRF, NF-κB and MAPK pathways(Fujigaki et al. (2006) J Biochem 139:655-662), activate indoleamine 2,3dioxygenase (IDO) which shifts tryptophan metabolism toward kynurenineand away from serotonin, thus reducing serotonin availability (Dantzeret al. (2008) Nat Rev Neurosci 9:46-56; Schwarcz et al. (2002) TheJournal of pharmacology and experimental therapeutics 303:1-10).

Kynurenine (converted to kynurenic acid in microglia) also inhibitsrelease of glutamate and, by extension, dopamine (Borland et al. (2004)J Neurochem 91:220-229). Other work on potential pathways leading todepression demonstrate that cytokine signals may significantly affectneural plasticity, triggering decreased neurotrophic support, decreasedneurogenesis, increased oxidative stress and even increased apoptosis inthe CNS (Buntinx et al. (2004) J Neurosci Res 76:834-845; Goshen et al.(2007) Psychoneuroendocrinology 32: 1106-1115; Koo et al. (2008)Proceedings of the National Academy of Sciences of the United States ofAmerica 105:751-756; Li et al. (2008) J Neurosci 28:5321-5330; McTigueet al. (2008) J Neurochem 107:1-19). Perhaps effects on neuralplasticity may explain why, in some disorders of the genitourinary tractsuch as interstitial cystitis, debilitating psychiatric effects canpersist long after any localized inflammation is measureable.

Finally, cytokines may have dramatic effects on thehypothalamic-pituitary-adrenal (HPA) axis (Goshen et al. (2008) MolPsychiatry 13:717-728) and dysregulation of the HPA-axis has beensuggested to underlie increased psychological stress levels inoveractive bladder and interstitial cystitis patients, at least thoseexposed to chronic early life stress (Taylor (2010) PNAS 107: 8507-8512.The contributions off these various pathways to the mood disordersexperienced by patients with diseases of the lower urinary tractrepresents important and exciting areas for exploration while offeringthe promise of targeted pharmacological interventions to alleviate thehigh morbidity and health-care costs associated with the mentalsuffering of urology patients.

In conclusion, this study has shown in an animal model that an acuteinsult in the bladder can trigger significant neuroinflammation in thehippocampus which brings about symptoms of depression. Moreover, theinflammation/depression responsive is dependent on activation of theNLRP3 inflammasome. Thus, this study proposes the first-ever causativeexplanation of the previously anecdotal link between benign bladderdisorders and mood disorders.

Materials and Methods for Examples 16-19

Animals

Animal protocols were approved by the Institutional Animal Care and UseCommittee at Duke University Medical Center and were performed inaccordance with the guidelines set forth in the Guide for the Care andUse of Laboratory Animals published by the National Institutes of Health(USA). Sprague Dawley Rats (female, ≈50 days of age, ≈200 g) werepurchased from Envigo (Indianapolis, Iowa) used in all experiments.Although in humans BOO occurs most frequently in males, the standard forBOO studies in rodents has long been the female rat. The primary reasonis the tortuosity of the male urethra which can lead to physical damage,and consequently inflammation, during catheterization. Other concernsare complications arising from ducts associated with the prostatic glandand seminal vesicles. Thus, male animals are contraindicated in studiesof BOO, particularly when examining inflammation and the results ofinflammation.

For most studies rats were assigned to 4 groups; 1) Control, 2) Sham, 3)BOO or 4) BOO+glyburide (Gly). An additional group (BOO+fluoxetine) wasused for behavior assays. For Sham and all BOO groups, animals wereanesthetized [ketamine hydrochloride (90 mg/kg), xylazine (10 mg/kg);i.p.] and a 1 mm o.d. catheter (P50 tubing) inserted transurethrally.BOO was created by urethral ligation over a 1 mm catheter. A 5-0 silksuture was passed around the urethra and tied securely for BOO andloosely for Sham. The catheter was removed and the abdominal wallclosed. For BOO+Gly, a subcutaneous pocket was made on the side of theneck and a single 50 mg, 21-day slow release pellet (Innovative Researchof America, Sarasota, Fla.) inserted and the incision closed. A newpellet was placed (contralateral side) after 21 days. Sides werealternated thereafter. No signs of urinary tract infection were everseen in any animal.

In the behavior assays, an additional group of BOO rats were providedwith fluoxetine (Sigma, St Louis, Mo.) in the drinking water (0.50mg/ml) for the last 4 weeks of the experiments. Twice weekly fluoxetinedose was adjusted to insure ≈20 mg/kg/day.

Evans Blue Assay

Rats were weighed and then injected (i.v. 3 ml/kg) with 2% Evan's bluedye in sterile saline by an investigator blinded to the groups (Belayevet al. (1996) Brain Res 739: 88-96). After one hour, rats wereeuthanized and transcardially perfused with cold PBS to removeintravascular dye. The brain was isolated and the hippocampus dissected,weighed and placed into formamide (0.25 ml) overnight (56° C.).Absorbance (620 nm) was measured and compared to a standard curve tocalculate pg Evans blue/μg tissue.

Immunocytochemistry and Quantitation of Microglia and Neurogenesis

Brains were fixed (10% neutral buffered formalin; 48 h, rt) and grosslycut (coronally) to the hippocampus before being embedded in paraffinwith the plane of the hippocampus on the block face. Citrate antigenretrieval was used prior to staining 10 μm coronal sections withanti-IbA1/AIF1 (1:500) (NBP2-19019; Novus Biologicals, Centennial,Colo.) or Anti-Ki67 (1:500) (ab15580; Abcam, Cambridge, Mass.) viastandardard methodology. IbA1 was visualized via HRP development(Vectastain ABC Kit; Vector, Burlingame, Calif.), biotinylated secondaryantibody provided). Ki-67 was visualized using a goat anti-rabbitsecondary antibody conjugated to Alexa Fluor 488 (111-545-144; JacksonImmunoResearch Labs, West Grove, Pa.). All slides were coverslippedusing Vectashield Antifade Mounting medium with DAPI (Vector,Burlingame, Calif.).

A Zeiss Axio Imager 2 microscope (Zeiss, Oberkochen, Germany) runningZen software (Zeiss) using the tiling and stitching feature was used toscan one entire hemisphere. Images were exported as TIFFs and importedinto NIS-Elements software (Nikon Co., Tokyo, Japan) then calibrated.Slides were quantitated by a researcher blinded to the groups. Formicroglia, we demarcated 600,000-700,000 μm² of the fascia dentata,counted the number of black/brown spots with two or greater associatedtendrils and calculated microglial density. For Ki-67⁺ cells wedemarcated the entire dentate gyrus, CA1, CA2 and CA3 regions,quantitated nuclei (DAPI⁺) that were Ki-67⁺ (green florescence) andcalculated the density.

Behavioral Assays

Open field—Rats are placed in an acrylic open-topped box (45 cm×45 cm×30cm; L×W×H) and video recorded for 10 min. The bottom is white while thesides are clear. The floor was divided into 16 equal squares. Afterrecording, the video is scored by individuals blinded to treatment andthe amount of time in which two or more of the rats paws are inside thecentral 4 squares (time in middle) recorded.

Sucrose preference—rats are simultaneously provided with bottlescontaining 2% sucrose or drinking water. The location of the bottles areswitched after 24 h. After 48 h the remaining liquid was measured andsucrose preference calculated as a percentage of the total liquidintake.

Statistical Analysis

Statistical assessments were conducted with Graph Pad In Stat Software(La Jolla, Calif.). ANOVA was used to examine differences across thefour groups, followed by a Student-Newman-Keuls post-hoc analysis thatenabled pairwise comparisons. To examine the effect of BOO oninflammation, bladder weight, neurogenesis and behavioral dysfunction inthe hippocampus, we compared control (and sham) group to the untreatedobstructed group. To examine whether treatment returned outcomes tobaseline, neurogenesis and behavioral dysfunction, we compared control(and sham) group to the treated obstructed group. Lastly, to examinewhether treatment ameliorated inflammation, we compared untreated andtreated obstructed groups. Results were considered statisticallysignificant if p<0.05.

Example 16: BOO Increased Bladder Weight and this was Partially Blockedby Inhibiting NLRP3

Bladders from the various groups indicated in FIG. 18 were weighed whenthey were removed for the various endpoints in this experiment. As shownin FIG. 18, there was no difference in bladder weight in sham-operatedrats compared to control. However, after 12 weeks of BOO bladder weightsincreased well over 10-fold. Glyburide suppresses this increase,although the values were considerably larger than controls.

Discussion:

At 12 weeks of BOO, bladder weight increased even in the presence ofglyburide, although the increase was attenuated in the drug-treatedanimals. The reason for the bladder weight gain in the glyburide-treatedrats was not directly examined, but weight gain is composed ofinflammation/edema and muscle hypertrophy and, while glyburide can beexpected to blunt the inflammation/edema it is less likely to do so formuscle hypertrophy caused by overuse of the detrusor muscle.

Example: 17 BOO Triggers NLRP3-Dependent Inflammation in the Hippocampus

To determine whether BOO triggers NLRP3-dependent inflammation in thehippocampus, BOO rats were treated with vehicle or glyburide andinflammation was assessed by the Evans blue assay as described in theMaterials and Methods section. As shown in FIG. 19, BOO triggered astatistically significant increase in the leakage of Evan's blue intothe hippocampus, indicating inflammation and disruption of the bloodbrain barrier. This leakage was reduced back to control levels byglyburide treatment. There was no difference between sham and control.To confirm this result, the density of microglia in the fascia dentatewas quantitated. Activated microglia in 10 μm sections of brain werestained for IbA1/AIF1 and then visualized and quantitated as describedin the Materials and Method section. FIG. 20 shows that there was nodifference in the sham-operated groups but a statistically significantincrease in the density of activated microglia in the hippocampusfollowing 12 weeks of BOO. Again this difference was reduced back tocontrol levels by glyburide.

Discussion:

Critical to the hypothesis was the detection of inflammation in thehippocampus, initially ascertained using the Evans blue assay whichmeasures the extravasation potential of the capillaries. When oneconsiders the blood vessels traversing the brain, this assay alsoequates to a measurement of the integrity of the blood brain barrier(Belayev et al. (1996) Brain Res 739: 88-96). Thus, after 12 weeks BOOhas precipitated a notable degradation of the blood brain barrier. Thepresence of inflammation suggested by the Evans blue assay was confirmedby the increase in activated microglia, the main drivers ofneuroinflammation and the cells in the brain possessing NLRP3 (alongwith astrocytes).

Importantly, both the Evan's blue extravasation and the increase inmicroglia were blocked by glyburide, indicating their absolutedependence on NLRP3. While that fact is irrefutable, it is somewhatunclear where, exactly, the glyburide is functioning; at the level ofthe bladder, the brain or both. Undoubtedly, glyburide is acting in thebladder urothelia (Hughes et al. (2016) J Urol 195: 1598-1605; Hughes etal. (2019) Am J Physiol-Renal 316: F113-F120; Lutolf et al. (2018)Neurourol Urodyn 37: 952-959), as there are several publications showingjust that. These urothelial effects also support the proposedinflammatory bladder-brain axis. Glyburide activity in the brain is notso clear. In the serum, glyburide is mostly albumin-bound and does notcross the blood brain barrier (Lahmann et al. (2015) LoS One 10:e0134476). However, in the event of a breakdown in the barrier, eventransiently, glyburide can cross into the brain (Stokum et al. (2017)Behav Brain Res 333: 43-53) where it could function to block NLRP3 inmicroglia (and perhaps astrocytes), minimize neuroinflammation andpreserve psychiatric health. Thus, the initial, major and perhaps onlyeffect of glyburide is in the bladder but, if its protective effect isoverwhelmed and there is breakdown of the blood brain barrier, it mayenter the brain and directly prevent a neuroinflammatory response.

Example 18: BOO Causes a Decrease in the Number of Proliferating Cellsin the Hippocampus

To determine if BOO causes a decrease in the number of proliferatingcells in the hippocampus, cells in 10 μm sections of brain were stainedfor Ki-67 and then visualized and quantitated as described in theMaterials and Method section. As shown in FIG. 21, there was astatistically significant decrease in the concentration of proliferatingcells (Ki-67⁺) in the hippocampus following BOO. The decrease wasblocked with concomitant administration of glyburide. There was nodifference between sham and control.

Discussion:

In the hippocampus BOO caused a statistically significant decrease inproliferating cells (Ki-67⁺). Decreases in neurogenesis, and moregenerally plasticity, in the hippocampus have been directly linked withneuroinflammation and depression (Liu et al. (2017) Neural Plast6871089), so given the neuroinflammation detected with the Evans blueand microglial activation assays, along with the signs of depressiondetected with the open field and sucrose preference assays, we feel thedifference in Ki-67⁺ cells likely reflect differences in neurogenesis.It is thought that these changes may lead to permanent, or at least longlasting, differences in cognitive function or mood that persists afterthe initiating stimulus is gone (in the case of BOO, after a TURP isperformed). This decrease was also blocked by glyburide demonstratingthe central role of NLRP3 and suggesting that NLRP3 inhibitors mayfurther help prevent BOO-induced neurodeterioration by suppressingnegative changes in neuroplasticity.

Example 19: BOO Rats Show NLRP-3 Dependent Signs of Depression; Anxietyand Anhedonia

Two different behavior assays that assess different signs associatedwith depression were performed. The open field test measures anxiety asa function of the rat's propensity to explore the middle region of asquare open field. Normal rats, being somewhat curious, will naturallyexplore this region while anxious rats refrain. The assay was performedas described in the Materials and Methods section and scored by ablinded investigator. As shown in FIG. 22A, BOO rats spent less thanhalf the time of the control and sham rats exploring the middle of thefield. Interestingly, glyburide restored this behavior demonstratingthat this anxious behavior is NLRP3-dependent. In addition, a separateset of BOO rats were given the antidepressant fluoxetine. Fluoxetinehelps to differentiate true depression-related behavior from sickbehavior, which is not affected by this antidepressant. As shown in FIG.22A, fluoxetine alleviated the depression and restored the exploratorybehavior of these rats back to levels not statistically different fromcontrol.

Next, the sucrose preference assay that assesses anhedonia or theinability to feel pleasure was performed. The assay was performed asdescribed in the Materials and Methods section. As shown in FIG. 22Bthere was a statistically significant decrease in the preference forsugar laden water in the BOO rats. Importantly, this preference wasrestored by treatment with glyburide. Fluoxetine also restored sucrosepreference back to levels not statistically different from control.There was no difference between sham and control.

Discussion:

Initial studies on the response of the innate immune system to BOOfocused on the local response in the bladder (Hughes et al. (2016) JUrol 195: 1598-1605; Hughes et al. (2019) Am J Physiol-Renal 316:F113-F120; Lutolf et al. (2018) Neurourol Urodyn 37: 952-959). Thosestudies found that BOO activates the NLRP3 inflammasome in the urotheliato initiate inflammation, fibrosis and denervation. In this study wehave greatly expanded that work to examine BOO-induced inflammation inthe brain and changes it may make in behavior. Initially, hippocampalinflammation was found after 6 weeks but differences in sucrosepreference at that time point were undetectable FIG. 23A and FIG. 23B,which necessitated performing this project after 12 weeks ofobstruction. Thus, inflammatory differences likely preceded developmentof anhedonia and anxiety by several weeks suggesting, not surprisingly,a sequential series of events in which inflammation precedes behavioralalterations.

The most important observation in this study was the behavioraldifferences in the BOO rats. Untreated, obstructed animals showedstatistically significant signs of anxiety and anhedonia, two coresymptoms of depression, and these behavioral differences were blocked byglyburide demonstrating the centrality of this inflammasome in thesemood changes. Importantly, the behavioral differences could also beprevented by the antidepressant fluoxetine demonstrating they are notdue simply to pain or “sick” behavior as fluoxetine would not beexpected to help in those situations.

One intriguing question that arises is the nature of theperipheral-to-central inflammatory signal. This is a hotly debated topicand currently three possible pathways are in vogue that are not mutuallyexclusive (Miller et al. (2016) Nat Rev Immunol 16: 22-34). The firstpathway, the humoral pathway, could result from cytokines producedlocally in the bladder travelling systemically via the vascular system,first to the blood brain barrier where they trigger breakdown, andsubsequently into the CNS where they initiate microglial activation andneuroinflammation. Given the breakdown of the blood brain barrier inthis study, this pathway is likely to contribute to BOO-inducedneuroinflammation. Another possibility is the neural pathway wheresensory input along afferent nerves carries a retrograde signal forinflammation back to the hippocampus where it is translates intocytokine production. Finally, a cellular pathway could involvetransmigration of immune cells activated in the bladder directly acrossthe blood brain barrier and into the brain vasculature and parenchyma.All three of these potential pathways warrant testing in future studies.

This study clearly shows that inflammatory injury to the bladder duringBOO causes central inflammation and mood disorders. It implies,therefore, that relieving the obstruction will relieve the mooddisorder. This work provides the first experimental animal data tyingbenign bladder dysfunction to mood disorders, and provides an excitingmechanism that might drive initiation and progression.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which thedisclosure pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference. In case of conflict, the presentspecification, including definitions, will control.

One skilled in the art will readily appreciate that the presentdisclosure is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentdisclosure is presently representative of embodiments, are exemplary,and are not intended as limitations on the scope of the invention.Changes therein and other uses will occur to those skilled in the artwhich are encompassed within the spirit of the disclosure as defined bythe scope of the claims.

1. A method of treating inflammation in the bladder in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of an inflammasome inhibitor.
 2. The method of claim 1,wherein the inflammation in the bladder is an acute inflammation or achronic inflammation.
 3. The method of claim 1, wherein the inflammationin the bladder is induced by a danger associated molecular pattern(DAMP) or a pathogen associated molecular pattern (PAMP).
 4. The methodof claim 3, wherein the PAMP is a fungus, bacteria, or virus.
 5. Themethod of claim 1, wherein the subject is a human.
 6. The method ofclaim 1, wherein the inflammasome inhibitor is an NLRP1 inflammasomeinhibitor, an NLRP3 inflammasome inhibitor, an NLRP6 inflammasomeinhibitor, an NLRP7 inflammasome inhibitor, an NLPR9 inflammasomeinhibitor, an NLRP12 inflammasome inhibitor, an NLRC4 inflammasomeinhibitor, or an AIM2 inflammasome inhibitor.
 7. The method of claim 1,wherein the inflammasome inhibitor is an NLRP3 inflammasome inhibitor.8. The method of claim 7, wherein the NLRP3 inflammasome inhibitor is aTXNIP inhibitor, ASC inhibitor, NEK7 inhibitor, Gasdermin D inhibitor,capspase-11 inhibitor, capsase-1 inhibitor, IL-1β inhibitor, IL-18inhibitor or combinations thereof.
 9. The method of claim 7, wherein theNLRP3 inflammasome inhibitor is glyburide.
 10. The method of claim 1,wherein the subject is diagnosed with diabetes, urinary tract infection,urinary frequency, fibrosis, bladder outlet obstruction, interstitialcystitis, CP-induced cystitis, depression, anxiety, neuroinflammation, agynecologic cancer, kidney stones, a pelvic inflammatory disorder,endometriosis, Chron's disease, diverticulitis, lupus, tuberculosis, andcombinations thereof.
 11. The method of claim 1, wherein the subject hadbeen exposed to chemotherapy, radiation, a catheter, or a urinary stent.12. A method of treating diabetic bladder dysfunction (DBD) in a subjectin need thereof, the method comprising administering to the subject atherapeutically effective amount of an inflammasome inhibitor.
 13. Themethod of claim 12, wherein the inflammasome inhibitor is an NLRP1inflammasome inhibitor, an NLRP3 inflammasome inhibitor, an NLRP6inflammasome inhibitor, an NLRP7 inflammasome inhibitor, an NLPR9inflammasome inhibitor, an NLRP12 inflammasome inhibitor, an NLRC4inflammasome inhibitor, or an AIM2 inflammasome inhibitor.
 14. Themethod of claim 13, wherein the inflammasome inhibitor is an NLRP3inflammasome inhibitor.
 15. The method of claim 14, wherein the NLRP3inflammasome inhibitor is a TXNIP inhibitor, ASC inhibitor, NEK7inhibitor, Gasdermin D inhibitor, capspase-11 inhibitor, capsase-1inhibitor, IL-1β inhibitor, IL-18 inhibitor or combinations thereof. 16.The method of claim 14, wherein the NLRP3 inflammasome inhibitor isglyburide.
 17. A method of treating or preventing a condition associatedwith neuroinflammation in a subject, the method comprising administeringa therapeutically effective amount of an inflammasome inhibitor.
 18. Themethod of claim 17, wherein the subject has been diagnosed withinterstitial cystitis, BOO, or DBD.
 19. The method of claim 17, whereinthe condition associated with neuroinflammation in a subject is a mooddisorder.
 20. The method of claim 17, the method further comprisingadministering a therapeutically effective amount of an antidepressantagent.